An in vitro method for inducing titan cells reveals novel features of yeast-to-titan switching in the human fungal pathogen Cryptococcus gattii.

Cryptococcosis is a potentially lethal fungal infection of humans caused by organisms within the Cryptococcus neoformans/gattii species complex. Whilst C. neoformans is a relatively common pathogen of immunocompromised individuals, C. gattii is capable of acting as a primary pathogen of immunocompetent individuals. Within the host, both species undergo morphogenesis to form titan cells: exceptionally large cells that are critical for disease establishment. To date, the induction, defining attributes, and underlying mechanism of titanisation have been mainly characterized in C. neoformans. Here, we report the serendipitous discovery of a simple and robust protocol for in vitro induction of titan cells in C. gattii. Using this in vitro approach, we reveal a remarkably high capacity for titanisation within C. gattii, especially in strains associated with the Pacific Northwest Outbreak, and characterise strain-specific differences within the clade. In particular, this approach demonstrates for the first time that cell size changes, DNA amplification, and budding are not always synchronous during titanisation. Interestingly, however, exhibition of these cell cycle phenotypes was correlated with genes associated with cell cycle progression including CDC11, CLN1, BUB2, and MCM6. Finally, our findings reveal exogenous p-Aminobenzoic acid to be a key inducer of titanisation in this organism. Consequently, this approach offers significant opportunities for future exploration of the underlying mechanism of titanisation in this genus.

Introduction

C. neoformans and C. gattii are two pathogenic species of Cryptococcus that cause invasive cryptococcosis in immunocompromised patients as well as immunocompetent individuals [1-3]. Upon inhalation into the lungs, Cryptococcus is exposed to a repertoire of hostile host factors (e.g., elevated temperature, nutrient deprivation, higher physiological CO2 and hypoxia), [4,5] which trigger adaptive phenotypes such as the formation of titan cells. Titan cell formation is a dramatic morphological change as cryptococcal haploid yeast cells (5–7μm) transform into enormous polyploid titan cells (50–100μm in diameter) [6,7]. This atypical morphotype is characterized by many attributes such as enlarged cell size, thicker cell wall, and altered capsule composition, which confer resistance to host immune defence and enhance survival in the host [8-10].

C. neoformans titan cells have been clinically observed [11,12], studied in vivo [6,7] and recently induced in vitro [13-15]. The discovery of in vitro induction protocols is considered a major breakthrough, as efforts employed towards understanding the biology and mechanism underlying titanisation were impeded by the ethical and technical challenges associated with using animal models. With the invention of in vitro induction models, key regulators of titan cell formation have been identified [16]. Exogenous factors including host-specific environmental conditions [such as physiological temperature (37°C) and CO2 level (5%) coupled with hypoxia, nutrient limitation etc.] and other cues (including serum) have been identified as essential signals, while low-density growth (via quorum sensing) serves as a regulator of titan cell formation [14,15,13]. Although many molecular details remain unclear, endogenous genetic regulators and transcription factors associated with titan cell formation include several members of the cAMP/PKA pathway (including Gpa1, Cac1, Ric8, Pka1 Rim101 and Usv101) [15,13,17]. Titan cell formation is also dependent on altered cell cycle progression, as cells undergo endoreduplication (replication of nuclear genome in the absence of mitosis) to form dramtically enlarged, polyploid cells [16]. During titanisation, C. neoformans yeast cells initially exhibit a stationary-phase growth during which DNA duplication occurs to form unbudded 2C/G2 arrested cells [18]. Thereafter, the cell cycle regulator Cln1 is suppressed, allowing DNA re-replication, creating polyploid titan cells. After titan cells have been formed, Cln1 is then upregulated in order to release cells from G2 arrest and enable them to reenter the cell cycle and produce daughter cells.

Although titanisation is thought to be a major virulence factor of C. gattii [19-21], the defining attributes and underlying mechanism of titanisation have thus far been mainly characterized in C. neoformans [22-24]. Here we describe a facile in vitro induction approach that reveals a novel strategy for titanisation in C. gattii. Specifically, this method reveals that the C. gattii/VGIIa strain R265 deviates from the usual synchronous occurrence of cell enlargement and polyploidization that occurs in C. neoformans. Instead, R265 delays DNA endoreduplication, growing to around 30μm as a haploid cell over the first 3 days of induction before endoreduplicating its DNA to become a uninucleate polyploid cell. Secondly, unlike C. neoformans, R265 titan cells produce daughter cells before reaching their critical cell size, after which they permanently cease budding unless they are exposed to conditions compatible with normal yeast growth. Although occurrence of these cell cycle phenotypes is asynchronous, these behaviors during titanisation are correlated with the expression levels of genes associated with cell cycle progression such as CLN1, CDC11, MCM6 and BUB2. By studying offspring from crosses between R265 and other C. gattii strains, we show that the propensity to titanise is most likely a highly polygenic trait and identify a number of hybrid strains in which in vitro titanisation levels approach 100%, opening the door to future molecular studies of this biological phenomenon.

Finally, we discovered that the widespread metabolite and UV-scavenger p-Aminobenzoic acid (pABA) is required for maximal titanisation in vitro, providing a molecular inroad to future investigations of the titanisation pathway.

Results

Titan cells are induced by growth in RPMI medium

While characterizing co-culture of C. gattii with an alveolar macrophage cell line (MH-S) at a low multiplicity of infection (MOI [5:1]), we noticed that exposure of low density R265 cultures to sterile RPMI growth media at 37°C in an atmosphere of 5% CO2 induced dramatic cell size increase within 24 hrs (Fig 1A and 1B). Although the phenotype was more prominent in the presence of serum supplemented-RPMI (median cell body size: 13.57μm vs 9.8μm, S1A Fig), the ability of serum-free RPMI to produce these enlarged cells provided the opportunity to probe the molecular basis of titanisation in this species using a simple, chemically-defined medium. Extending the incubation period to seven days enabled these cells to achieve cell bodies of up to 30μm in diameter even in the absence of serum, resulting in a population with a median size significantly higher than yeast cells grown in YPD at 37°C in an atmosphere of 5% CO2 for 7 days [median size: 13.7μm (5.1–29.8) vs 10.17μm (3.00–19.11) (p<0.0001)] (Fig 1C and 1E [⍺ = cell body diameter], S1B Fig). Notably, the pH of both media remained close to neutral throughout the assay, ruling out a secondary impact on titan cell formation as a result of pH stress (S1C Fig).

Fig 1

C. gattii (R265) exhibits cell body and capsule enlargement in response to growth in RPMI.

A) Micrographs of R265 cells grown in YPD overnight at 25°C in atmospheric conditions (left panel), or at 37°C in an atmosphere of 5% CO2 either in YPD (middle panel) or RPMI (right panel). Scale bar = 15μm. B) Total cell diameter (capsule included), C) cell body diameter (percentages represent percentage of cells more than 10μm in diameter) and D) capsule thickness of R265 cells all significantly increase after growth in RPMI at 37°C in 5% CO2. The graphs represent at least 3 biological experimental repeats, and Kruskal-Wallis test was used to determine significance where **** = p<0.0001 and ns = p>0.05. E) Cellular morphology of R265 enlarged cells: cell body diameter (⍺) and capsule thickness (ß). Scale bar = 15μm.

The cryptococcal yeast-titan transition occurs concomitantly with increasing capsule thickness [19]. Therefore, we characterized the capsule size (Fig 1D and 1E [ß = capsule size]) of our in vitro-generated R265 enlarged cells. Relative to the YPD-grown capsule thickness, R265 enlarged cells demonstrated significantly thicker capsule [median capsule thickness: 10.28μm (0.34–22.9) vs 1.00μm (0.2–3.1), p<0.0001]. Although the cell body size progressively increased with induction time from 24 hr to 7 days (Fig 1C), the capsule size of the enlarged cells reached a plateau at 24 hours and remained at a median size of 10.33μm (1.0–22.9) for the remaining six days of the assay (Fig 1D). This suggests that the enlarged cells achieve their maximum capsule size much earlier than their maximum cell body size.

Titan cells exhibit altered cell wall composition [13,15,25,26]. Consequently, we characterized the cell wall of R265 in vitro-derived enlarged cells by staining for chitin with calcofluor white (CFW) [15]. In line with previous studies, we observed a significant increase (p<0.0001) in the chitin content of enlarged induced cells via flow cytometry that was also evident via fluorescence microscopy (CFW, Fig 2A, 2B and 2C). In addition to displaying a titan-like capsule, cell body and cell wall, C. gattii (R265) enlarged cells bear a single large vacuole occupying almost the entire cytoplasmic space (Fig 2D), a characteristic previously described in both in vivo and in vitro-derived C. neoformans titan cells [7,13].

Fig 2

The cell wall chitin content and ploidy of enlarged cells are typical of titan cells.

In vitro RPMI-generated enlarged cells displayed a significantly higher chitin level relative to YPD grown R265 cells, as indicated by the calcofluor white (CFW) fluorescence intensity shown using microscopy imaging in (A), via flow cytometry in (B) and graphically in (C, Median Fluorescence Intensity, MFI) (p<0.0001). Scale bar = 15μm. Statistical significance was confirmed by Two-tailed t-test. D) Micrograph showing single large central vacuole of in vitro-generated enlarged cells. E) Ploidy measurement of RPMI-generated enlarged cells based on flow cytometry analysis of DAPI staining. YPD grown cells (red) gated as 1C, 2C (haploid) were used as a control to determine ploidy (DNA content) of enlarged cells after 24 hr (blue) and 7 days (orange) of induction. F) Micrographs showing the uninucleate nature of R265 titan cells upon staining with DAPI to visualize the nucleus. Scale bar = 10μm.

In addition to morphological changes, the yeast-titan transition in C. neoformans involves a switch from a haploid to highly polyploid state [6,7,13,15]. Thus, we evaluated the ploidy of RPMI in vitro-generated C. gattii enlarged cells. Whilst YPD grown yeast cells typically displayed a mix of cells with 1C or 2C DNA content (depending on which phase of the cell cycle they are in), RPMI-induced cells displayed DNA content ranging up to 16C after 7 days of induction (Fig 2E). By DAPI staining the nucleus and visualizing by microscopy, we confirmed the enlarged cells were uninucleate (Fig 2F). Thus, R265 titan-induced cells exhibit all the key features of bona fide titan cells: cell enlargement, a large vacuole, altered cell wall composition and high ploidy. Interestingly, however, we noted that in the case of R265, cell enlargement and increased DNA content do not necessarily occur at the same time. Indeed, R265 cells achieve significant cell body enlargement within 24hrs, but DNA content does not exceed 2C until much later (Fig 2E). Taken together, the structural attributes exhibited by the in vitro RPMI-induced titan cells are typical of true titan cells.

Titan cell formation is inversely correlated to cell density

Previous work in C. neoformans has shown that titan cells are preferentially produced at low cell density [7,14,15,27]. We tested whether the same was true of R265 by growing yeast cells in RPMI at five decreasing inoculum concentrations (106, 5x105, 5x104, 5x103 and 103 cells/mL) and incubating for 24hr at 37°C in 5% CO2. The formation of titan cells was maximal [87.9% titan cells (>10μm)] at an initial inoculum of 5x103 cells/mL, producing cells with a median cell body size of 14.8μm (4.2–26.28) (Fig 3A). The percentage of titan cells decreased with increasing cell densities, indicating that in C. gattii, as in C. neoformans, titan induction occurs primarily in low density cultures.

Fig 3

Effect of cell density and environmental conditions on R265 titan cell formation.

A) The effect of cell density on cell enlargement was determined by growing R265 cells in titan-inducing conditions at varying concentrations (between 106 and 103 cells/mL, as indicated) and cell size being measured 72 hrs later (percentages indicate proportion of the population >10 μm). B) The role of the quorum sensing peptide, Qsp1 (on titan cell formation) was determined by growing R265 yeast cells in RPMI supplemented with the intact and scrambled peptide (control) at 37°C with 5% CO2 and measuring the cell body size after 72 hr incubation. Microscopy images illustrating the morphological differences in titan cell production between these three conditions (RPMI, intact Qsp1 and scrambled Qsp1) are also shown. Maximum cell enlargement capacity (C), budding index (D) and ploidy (E) of R265 cells in RPMI at 106 cells/mL (high density growth) at 37°C with 5% CO2 for 24 hr and 7 days. Budding index was expressed as percentage of budded cells per total number of cells. At least >3000 cells were analysed for each sample from two independent repeats and significance was determined by one-way ANOVA (**** = p<0.0001). (E) Cells recovered from 7 day high-density induction cultures were analysed for ploidy (orange) which was consistent with a 1C and 2C haploid DNA content as found for the YPD grown cells (red). F) Microscopy images depicting the budding index of cells grown in YPD (left panel), RPMI for 24hr (middle panel) and RPMI for 7days (right panel). Scale bar: 10μm. G) Impact of temperature and 5% CO2 growth: cell body diameter of R265 cells after 24hrs of growth in RPMI with 5% CO2 at 37°C or 30°C. The graphs represent at least 3 biological experimental repeats, and Kruskal-Wallis test was used to determine significance where **** = p<0.0001.

Quorum sensing effect

The density dependence of titanisation is suggestive of a quorum sensing effect. Consequently, we tested the impact of the putative cryptococcal quorum sensing molecule Qsp1, a short peptide previously reported to inhibit titanisation in C. neoformans cells in vitro [14,15]. By adding the Qsp1 peptide (NFGAPGGAYPW) at 20μM to RPMI, both the median cell body size of the R265 cells and the proportion of titan cells was significantly reduced [median cell size: 13.35μm vs 11.74μm (p<0.0001); percentage titan cells: 88.3% vs 75.8%]. In contrast, a ‘sequence-scrambled’ Qsp1 peptide (AYAPWFGNPG) had no effect (Fig 3B). Interstingly, the active peptide also impacted capsule growth by reducing capsule size of the titan induced cells significantly (see micrograph images of Fig 3B). Taken together, this indicates that titan cell formation in C. gattii is modulated by quorum sensing via the Qsp1 pathway, as previously described in C. neoformans [14,15].

High density growth in RPMI produces large, haploid, non-titan cells

The impact of culture density led us to ask what happens to R265 in non-titanising (high density) conditions. To answer this, we inoculated R265 cells in RPMI at 106 cells/mL and measured cell body size, capsule size, and ploidy over 7 days. At high density, there was a statistically significant increase in cell body size from 24 hr to 7days (median size: 9.0μm vs 10.5μm, p<0.0001) (Fig 3C), but cells did not become the very large titans that appear in low density cultures (Fig 1C). At both time points in these high-density conditions, cell enlargement typically did not surpass 15μm, with 0.1% (4/4302) and 1.3% (46/3489) of cells scoring >15μm at 24 hr and 7 days respectively (Fig 3C). The 7-day cultures were fully growth-arrested, as evidenced by their very low budding index (the number of mother cells producing buds) of 2.1% (67/3122) (Fig 3D and 3F). These 7-day high-density cells did produce dramatic capsule (Fig 3F), consistent with the known effects of RPMI media and CO2, which have been employed for capsule induction in Cryptococcus [19,27-30]. Interestingly, unlike cells grown in low-density titan-inducing conditions, cells grown at high density remained haploid over the entire 7 day period (Fig 3E). We conclude that high-density long-term culture in RPMI induces a degree of cell enlargement in R265, but these large cells are distinct from the true titan cells that appear in low density culture.

Impact of temperature and CO2 on titan cell formation

Cryptococcus responds to human physiological temperature (37°C) by exhibiting a variety of morphological changes including capsule elaboration, cell body enlargement and cell shape alteration [13,30,31]. Hence, we investigated the role of temperature on R265 yeast-titan cell transformation by comparing cells grown in the presence of 5% CO2 at either 37°C or 30°C. At 30°C incubation, no titan cells were generated and the median cell body size [5.03μm (1.04–12.85)] was significantly lower than at 37°C [11.17μm (4.04–28.6)]. A similar trend was observed for the proportion of titan cells (13.8% vs 2.2%) (Fig 3G). Thus, elevated temperature was essential to produce titan cells in our in vitro protocol.

The most dramatic biological response of Cryptococcus to CO2 is capsule biosynthesis which occurs concurrently with cell body enlargement [19,29]. Consequently, we compared cells grown at 37°C in 5% CO2 with those grown under normal atmospheric conditions at 37°C. Relative to growth in 5% CO2, the proliferation of R265 was significantly inhibited in ambient atmosphere [Mean CFU: CO2 = 28.5 x105 cells/mL vs CO2- free 0.29 x105 cells/mL; (p<0.001)] and no titan cells were observed in this condition (S2A Fig). Thus, both elevated temperature and high CO2 are required for in vitro titan cell formation.

In R265, cell enlargement is asynchronous with ploidy

To characterise the kinetics of cell size and ploidy changes more fully, we carried out a detailed time course analysis of R265 cells over a period of three weeks. By day 3 of induction, R265 cells showed significantly enlarged cell body diameter as compared with non-induced (YPD grown) cells [median size: 13.7μm (5.1–29.8) vs 6.5μm (4.9–8.89); (p<0.0001)] with 81.5% (5241/6431) of cells larger than 10μm (Fig 4A). Despite this size increase, for the first three days all cells were 1C or 2C (reflecting a haploid cell cycle) (Fig 4B). The population reached the maximum cell size on day 5, and from day 5 to day 7, there was no change in the size of induced cells [median size: 14.9μm at day 5 and 14.7μm at day 7 (p = 0.7656)]. However, the ploidy of these cells increased, with tetraploid (4C) cells apparent at day 5 and cells exhibiting DNA content of up to 16C present by day 7. In parallel, although the maximum size of induced cells no longer increased at this late stage of incubation, the proportion of the population with a large cell phenotype rose from 92.8% to 99.3% (p<0.0001) (Fig 4A). Thus, it appears that: a) size increase and ploidy increase are separable phenotypes during titanisation of R265, b) true titan cells (large and polyploid) appear only after around 5 days of induction and c) their maximum cell size is achieved rapidly during titanisation, but the proportion of cells adopting this fate rises steadily over a long time period.

Fig 4

Cell enlargement, polyploidization and budding occur at different periods during titan induction in R265.

A) Cell body diameter of R265 titan cells over prolonged induction (24 hr to 21 days) as compared to YPD grown cells. Enlarged cells were recovered at different time points, fixed and measured. The error bars represent median of 3 biological repeats, where **** = p<0.0001. B) Samples from all the induction time points were fixed, DAPI stained and analysed for ploidy by flow cytometry with reference to YPD grown cells, which were used to gate for 1C and 2C DNA content. C) Budding index of cells recovered from YPD and titan inducing condition. Budding index was expressed as percentage of budded cells per total number of cells. At least 3000 cells were analysed for each sample (the graph represents three biological repeats and significance was confirmed by one-way ANOVA, where **** = p<0.0001 D) Microscopy images showing the budding nature of cells obtained from YPD or at various timepoints after titan induction. Scale bar: 15μm.

The polyploid titan cells are unbudded

Within our in vitro model, after 3 days of induction, the R265 titan cells completely stop budding despite being polyploid (Fig 4C and 4D). This suggests that budding and DNA increase are decoupled, consistent with endoreduplication. During the first 3 days, budding index fell from 44.4% (2552/5749 cells) at 24 hr to 2.8% (26/933) by day 3 and 0% (0/2971) by the fifth day of induction (Fig 4C). Hence, we termed the period before 3 days of induction as the budded phase and the later period as the unbudded phase. During the budded phase, cells predominantly exhibited a 1C DNA content (Fig 4B). However, during the unbudded phase (3 to 7 days) cells rapidly became polyploid, with DNA content rising from 2C to 4C to 16C (Fig 4B). The 1C DNA content during the budded phase, coupled with cell enlargement, suggests that the cells spend longer in the G1 phase of their cell cycle during titan induction.

Unbudded titan cells are viable and metabolically active

The unbudded state of C. gattii (R265) titan cells prompted us to investigate whether this cell cycle arrest occurs as a consequence of nutrient deprivation. By filtering 7 day old titan-induced cultures, we obtained the largest (>20μm) titan cells and re-cultured them on a rotary wheel at 20 rpm for i) 2 hr or ii) overnight at 25°C in YPD broth. These cells remained unbudded during the first 2 hrs (the generation period of Cryptococcus yeast) (Fig 5A) but after overnight incubation produced daughter cells (Fig 5B and 5C) resembling YPD-grown yeast cells in size and morphology (round-shaped with small capsule size) (Fig 5D). Time-lapse microscopy revealed that recovered titan cells start budding after 2 hours (S1 Movie). The active proliferation in YPD (nutrient rich media) of the unbudded titan cells suggest that their exhibition of growth arrest is due to nutrient depletion after the long incubation. In line with this, proliferation could also be observed when these unbudded titan cells were re-cultured in fresh RPMI (S2B Fig).

Fig 5

Characterization of daughter cells and metabolic state of R265 titan cells.

7 day old R265 titan cells were purified using a 20μm cell strainer (A) and then B) re-cultured overnight in YPD to induce budding. C) After 24hrs, daughter cells of R265 titan cells were isolated by filtration of the titan culture (through a >15μm cell strainer) and microscopically compared with D) YPD grown yeast R265 cells (control). E) Microscopy images demonstrating the metabolic activity titan cells from 7 days old cultured (fresh vs heat-killed) and YPD grown yeast cells (fresh vs heat-killed). The cells were stained with the metabolic reporter dye Fun-1 which is converted from yellow-green to orange-red by metabolically active cells. Scale bar = 10μm. F) DNA content of titan-derived daughter cells (Blue) cells as compared to YPD grown (red) and >20μm R265 titan cells (brown). G) Size distribution of daughter cells (blue), YPD grown (red) and >20μm R265 titan cells (brown).

We tested whether unbudded R265 titan cells are quiescent, but metabolically active, by using the dye Fun-1, whose emission wavelength is converted from yellow-green to orange-red colour by metabolically active cells. Unbudded titan cells were able to convert the FUN-1 dye in a manner that was comparable to actively-growing yeast cells (Fig 5E). Thus, both timelapse imaging and metabolic profiling indicates that titan cells remain viable and metabolically active.

Titan cells produce polyploid, yeast-sized, daughter cells

To investigate if the cellular similarities observed between titan-derived daughter cells and yeast cells extend to their ploidy, we characterized the DNA content of daughter cells relative to their highly polyploid titan mother cells (>20μm) and haploid yeast cells. Despite having the cellular properties of yeast cells (Fig 5C and 5G), titan-derived daughter cells exhibited a higher DNA content than normal yeast, with most cells displaying either diploid (2C) or polyploid (>4C) DNA content (Figs 5F and S3A shows gating strategy). Taken together, the delayed DNA replication of R265 titan cells (Fig 4B) coupled with the production of polyploid daughter cells (up to 8C) (Fig 4E) is indicative of high ploidy elasticity in C. gattii titan cells.

By re-culturing titan-derived daughter cells in titan inducing condition, we assessed the ability of these ‘second generation’ cells to return to a titan state. Unlike the ‘original’ titan cells, these titan-derived daughters increased their DNA content within 24 hours, achieving genome sizes of 16C and 32C at 24hr and 7 days respectively (S3E Fig). As with the original mother titan cells, titan-induced daughter cells underwent budding during the early induction period but formed unbudded titan cells by day 7 of induction (S3B and S3C Fig).

The titan cell cycle phenotypes are correlated with the expression of genes involved in cell cycle progression

The manner in which R265 progressively exhibits cell-cycle-associated phenotypes (cell enlargement, budding, DNA replication and finally growth arrest) to form unbudded polyploid titan cells led us to question the underlying cell cycle timing. We extracted RNA from R265 titan induced cells for 24 hr, 3 days, 5 days and 7 days of our in vitro protocol and then investigated the expression of a panel of cell cycle markers via quantitative RT-PCR (S1 Table).

In line with the phenotypic changes that we observe during titan cell formation, the cell cycle markers we examined showed a clear shift from budding and mitosis to DNA replication and eventually cell-cycle arrest (Fig 6). Thus, the CDC11 gene, encoding a septin protein involved in bud formation, was highly expressed in YPD grown cells and for the first 24hrs of induction, but downregulated in the "unbudded phase" timepoints (Fig 6A). The expression of the G1 cyclin CLN1 similarly was reduced at day 3 compared to 24 hrs, while expression was somewhat restored by day 5 (Fig 6B). In contrast, MCM6 and BUB2, associated with DNA replication and G2 arrest respectively, both peaked at 7 days where the titan cells exhibit maximum polyploidy and remain unbudded (Fig 6C and 6D).

Fig 6

Relative transcription of R265 cell cycle marker genes during titanisation.

Quantitative expression analysis of four different cell cycle associated genes in R265 grown in either YPD (control) or titanising conditions for the indicated time points. Expression is shown relative to the housekeeping gene GAPDH. Genes quantified were A) CDC11 (CNBG_5339), involved in bud formation; B) CLN1 (CNBG_4803), associated with balancing cell division and DNA replication [18]; C) MCM6 (CNBG_5506), involved in DNA replication; and D) BUB2 (CNBG_4446), involved in G2 arrest. The graphs represent 3 biological repeats (3 technical replicates each), with error bars depicting the standard deviation of delta-delta C values.

P-Aminobenzoic acid is a major trigger of titanisation in RPMI

While comparing the titan induction capacity of RPMI [RPMI Medium 1640 (1X)] against Dulbecco’s modified eagle medium (DMEM), we noticed that the full titan cell phenotype emerged in RPMI but not DMEM after 24 hrs of induction. Notably, cells enlarged in both media, but cells grown in DMEM never achieved the same size as those in RPMI and did not become polyploid. Since RPMI and DMEM media have a close chemical composition, we took advantage of this similarity and sequentially supplemented DMEM with RPMI-specific compounds with the aim of identifying an RPMI-specific factor that triggers titanisation in R265 cells. To more clearly distinguish between conditions, we used a higher threshold for titan cells in this assay (15 μm rather than 10 μm). After 24hrs of induction, 13.8% of cells in RPMI had a cell body size above 15 μm, whilst no cells of this size appeared in DMEM [median cell body size: 12.3 μm (4.1–28.8) vs 9.9 μm (4.5–14.9)] (Fig 7A). By 3 days of incubation, larger cells had appeared within the DMEM condition, but at a rate approximately three-fold lower than for RPMI (Fig 7B).

Fig 7

The effect of RPMI-specific compounds on R265 titan cell formation.

A) RPMI-specific amino acids (L-Glutamic acid, L-Aspartic acid, L-Arginine, L-Glutathione, L-Asparagine and L-Proline) were added to DMEM either at the concentration used in RPMI (‘low conc.’) or two-fold higher (‘high conc.’) and tested for their capacity to trigger titan cell formation after 24 hr induction at 37°C in 5% CO2. B) RPMI-specific compounds (Vitamin B12, Biotin, and para aminobenzoic acid (pABA)) were supplemented to DMEM at the levels present in RPMI and then evaluated for their capacity to induce titan cell formation after 3 days incubation at 37°C in 5% CO2. The graphs are representation of 3 biological repeats and statistical significance was determined by Kruskal-Wallis test, where **** = p<0.0001.

RPMI differs from DMEM in its amino acid composition and so we first supplemented DMEM with ‘RPMI-specific’ amino acids (L-Glutamic acid, L-Aspartic acid, L-Arginine, L-Glutathione, L-Asparagine and L-Proline). However, none of these conditions were sufficient to confer titan-inducing capacity to DMEM, even when supplemented at twice their normal concentration (Fig 7). We also increased the glucose concentration of DMEM from 1000mg/L to 2000 mg/L (RPMI concentration) but saw no significant impact on titan production (S1D Fig). Based on these results, we conclude that amino acid availability and glucose concentration are not the trigger for R265 titanisation.

We continued by testing three additional compounds that are present in RPMI but absent from DMEM: Vitamin B12, Biotin, and para-aminobenzoic acid (pABA). Whilst vitamin B12 or biotin supplementation into DMEM had relatively little effect, addition of pABA significantly increased the production of titan cells from 13.7% to 29.0% (median cell diameter = 11.94μm vs 12.82 μm, p <0.0001) (Fig 7B). Consequently, pABA appears to be a major trigger for titan cell formation in RPMI.

Finally, we noted that supplemental iron (in the form of Iron (III) nitrate nonahydrate-Fe (NO3)3.9H2O) is present in DMEM but absent from RPMI. We therefore tested whether iron availability may actively inhibit titanisation. Indeed, adding iron to RPMI (Fe (NO3)3.9H2O+RPMI) slightly reduced the induction capacity of RPMI by ~4%. This was not surprising as iron limitation induces titan cell formation in C. neoformans [14]. In summary, therefore, the efficient induction of titan cells in RPMI likely results from the combined presence of pABA and the absence of supplementary iron.

Strain specificity

There is considerable evolutionary divergence between clades within the Cryptococcus genus and indeed the nomenclature of this group is rapidly changing in recognition of potential cryptic species [32]. To begin to assess variation in titanisation capacity, we screened 42 different cryptococcal isolates comprising 32 C. gattii species complex strains (VGI–VGIV), 8 C. neoformans strains (VNI and VNII), and 2 C. deneoformans strains (VNIV) for their capacity to form titan cells in our in vitro protocol (3 days incubation in RPMI with 5% CO2 at 37°C). Overall, the capacity to form titan cells in C. gattii strains was significantly higher than either C. neoformans or C. deneoformans. All 32 C. gattii strains produced titan cells, with an average of 60% titan cells at the end of the assay (Table 1). In contrast, only 4/8 (C. neoformans) and 0/2 (C. deneoformans) strains showed any level of titanisation (Table 1). Within the C. gattii species complex, VGII genotype strains (C. deuterogattii) displayed the highest titanisation capacity (averaging 80.0%) while VGIII (C. gattii) scored the lowest at 37.9% (Table 1).

Table 1

Capacity to form titan cells across cryptococcal isolates.

Percentage of titan cells was determined based on capacity to enlarge >10μm (S4 Fig) and having >2C ploidy (S5 Fig).

Species/strain	Genotype	Median size [range] (μm)	% Titan cells
C. gattii
WM265	VGI	10.9 [4.1–28.4]	62.7
WM179	VGI	11.3 [4.1–25.0]	67
CBS8755	VGI	11.0 [4.6–17.7]	62.8
C384	VGI	10.0 [4.2–27.9]	50.1
NIH312	VGI	8.0 [4.1–24.3]	29.3
B4546	VGI	8.9 [4.5–26.2]	35.3
EJB11	VGI	10.8 [5.3–25.6]	65.5
MMCO8-897	VGI	6.7 [4.4–16.3]	17.5
CBS1508	VGI	9.0 [4.7–17.4]	27.3
CBC1873	VGI	11.6 [5.8–22.7]	62.3
	Av. of VGI	9.82	48.0
R265	VGIIa	13.4 [5.1–29.6]	79.7
CDDR271	VGIIa	12.9 [4.4–26.5]	84.7
ENV152	VGIIa	10.6 [5.5–28.0]	54.8
CDCF2866	VGIIa	11.3 [4.6–19.9]	89.1
ICB180	VGII	12.7 [3.4–26.3]	82.6
CBS10089	VGII	13.3 [5.6–25.1]	89.1
CDCR272	VGIIb	13.5 [6.8–39.2]	90
B7735	VGIIb	12.9 [5.5–26.3]	86.4
EJB18	VGIIc	11.3 [4.7–19.5]	75.6
EJB52	VGIIc	11.1 [4.8–19.5]	68.3
	Av. of VGII	12.3	80.0
CBS6955	VGIII	8.24 [4.34–17.9]	66.3
CBS6993	VGIII	9.9 [4.8–22.6]	45.7
CBS1622	VGIII	5.4 [3.3–10.9]	4.4
WM1243	VGIII	10.1 [4.6–22.4]	50.7
B13C	VGIII	10.0 [4.4–23.5]	50
CA2350	VGIII	8.8 [4.7–21.7]	32.7
CA1227	VGIII	7.1 [4.4–15.2]	15.4
	Av. of VGIII	9.1	37.9
WM779	VGIV	11.8 [4.2–25.5]	66.5
CBS1010	VGIV	12.6 [4.3–22.2]	80
B5742	VGIV	8.9 [4.6–21.8]	37.3
B5748	VGIV	10.7 [4.8–22.9]	61.9
CBS10101	VGIV	10.9 [4.4–19.6]	62.7
	Av. of VGIV	12.2	61.7
All C. gattii (Average)		10.75	60
C. neoformans
H99	VNI	7.7 [3.4–20.1]	21.3
Zc1	VNI	7.6 [4.46–15.9]	5.8
Zc8	VNI	12.4 [5.4–20.7]	76.1
Zc12	VNI	4.1 [2.1–7.0]	0
CBS8336	VNI	9.6 [4.6–24.8]	39.9
125.91	VNI	7.2 [4.6–16.3]	7.9
	Av. of VNI	8.1	23.8
Tu_406_1	VNII	10.0 [4.9–18.5]	0
HamdanC3’1	VNII	9.3 [4.5–22.1]	0
	Av. of VNII	9.6	0
All C. neoformans (Average)		9.7	18.8
(average)
B3501	VNIV	7.2 [4.5–17.43]	0
CBS6995	VNIV	4.5 [3.4–11.1]	0
	Av. of VNIV	5.85	0
All C. deneoformans (Average)		5.85	14.2

Titanisation in C. gattii is a polygenic trait

Several genetic regulators have been implicated in the control of titanisation in C. neoformans [6,7,13,15,24]. The interaction between these (nuclear genome-encoded) genetic regulators and mitochondrial activity has been proposed [16]. Consequently, we exploited a collection of parent/progeny crosses that we generated as part of an earlier study, and for which mitochondrial genotype is known [33], to begin to investigate the genetic control of titanisation in C. gattii. Mitochondrial genotype and inheritance were confirmed by the expression of ATP6 gene (encoded by the mitochondrial genome) [33].

Firstly, we investigated a cross between C. gattii R265 and C. gattii LA584, a strain that belongs to the same VGII group as R265 but which shows a significantly lower capacity to titanise in our in vitro conditions (Fig 8A). The progeny from this cross showed considerable variation in titanisation capacity, with only one (Alg30) exceeding the titanisation capacity of R265. Ploidy measurement of parents/progeny based on flow cytometry analysis of DAPI staining for DNA content is shown in S6A Fig. Notably, there was no correlation between titanisation capacity of individual progeny and the mitochondrial genotype (R265 or LA584) that they had inherited, suggesting that mitochondrial genotype is not a major driver of this phenotype.

Fig 8

Capacity to form titan cells of C. gattii progeny arising from two crosses.

(A) Titanisation pattern following three days of induction for R265 (VGII) x LA584 (VGII) and 13 progeny (Alg23-Alg35) arising from this cross [33]. (B) Titanisation pattern following three days of induction of R265 (VGII) x B4564 (VGIII) and 18 of the progeny (P1-P18) arising from this cross.

We then turned our attention to a cross between R265 and a more distantly related strain, B4564 (VGIII), which shows relatively low levels of titanisation. In this outgroup cross, a 100% inheritance of the mitochondrial genome from B4564 (MATa) was confirmed in 18 progeny [33]. Despite this uniparental mitochondrial inheritance, all the progeny exhibit a significantly higher capacity for titanization than B4564 by both size and ploidy (Figs 8B and S6B). Furthermore, 12/18 progeny showed a higher capacity for titanisation than either parent (p<0.0001) with 5 progeny (P1, P5, P7, P8, P9 and P18) showing a remarkable >95% titan cell population by the end of the assay. Together, these data suggest that the nuclear genome, and not the mitochondrial genome, is the major source of variation in the capacity of different isolates to form titan cells.

Discussion

Cryptococcus adaptation to the host environment is accompanied by phenotypic, metabolic and genetic alterations that are essential for pathogenicity [13,34-38]. Typically measuring 5–7 μm, the cryptococcal yeast cell can undergo a morphological switch in the lungs to form enlarged polyploid titan cells, which have been studied in vivo [6,7] and recently induced in vitro [13-15].

Here we report a new in vitro model for induction of bone fide titan cells. Our protocol is a one-step incubation of cryptococcal cells in serum-free RPMI media with 5% CO2 at 37°C and is therefore highly amenable to high-throughput screens.

Using our in vitro protocol, we conducted a detailed analysis of titanisation in the C. gattii strain R265 (VGIIa). We showed that R265 titan cells induced in vitro possess enlarged cell body size, a large central vacuole, thick capsule and cell wall, and were polyploid after 5 days. Consequently, they exhibit all the hallmarks of true titans produced during mammalian infection. Unlike other cryptococcal strains, however, R265 shows a separation between size and ploidy increase. We suggest that the asynchronous progression of these two events may be due to R265 undergoing cell size increase without passage through the cell cycle and then subsequently switching to DNA replication once the critical volume has been attained.

The lack of synchronisation between cell enlargement and DNA replication is a major difference between R265 titan cells and other well-studied Cryptococcus titan cells [6,7,13,14,16,24,27]. We suggest that the asynchronous progression of these two events is due to the prolonged time the titan cells spend in the G1 phase and/or cell cycle arrest at the G1/S checkpoint. This is supported by the ploidy of the 24 hr induction samples, where the vast majority of cells show a 1C DNA content. In yeast, a late G1 cell cycle arrest, known as “Start”, is part of the core cellular response to stress [39,40]. In R265 we observe that cell enlargement occurs before polyploidization during the early period of induction, so that the enlarged cells remain as 1C haploid yeast cells for the first two days of induction. By day 3 of induction, the enlarged cells begin to duplicate their DNA content to at least 2C but almost completely stop budding at the same time [budding index: 2.8% (26/933)] (Fig 4C). This leads to a major distinction between the species, in that C. neoformans titan cells can produce daughter cells within our in vitro titan induction model (S7 Fig) whilst C. gattii R265 titans do not. We note that this observation may be of value in studying cell cycle dynamics in Cryptococcus species (particularly C. gattii), since producing synchronised cryptococcal populations for such investigations has previously been methodologically challenging.

To explain the unbudded phase of the induced cells, we attempt to correlate this phenotype with cell cycle progression with reference to C. neoformans. In C. neoformans, large unbudded G2 cells have been shown to emerge during a stationary growth phase [41]. Recently, while scrutinizing the cell cycle regulation of titan cells in C. neoformans, Altamirano et al. [19] described a two-step process of titanisation: a) typically-sized cells duplicate DNA to 2C and arrest in G2 as unbudded cells; and b) then the cells are released (by the combined influence of the cell cycle gene Cyclin Cln1 and “stress signals”) to form polyploid titan cells. Contrary to this phenomenon, R265 cells exhibit an actively budding phase in the early period of induction where the majority of the cells display 1C DNA content consistent with G1 and then undergo DNA replication to form G2 arrested unbudded polyploid titan cells at the later time point (day 5 onwards) where cells with 1C DNA content are absent (Fig 4B). In agreement with this observation, we observed that the transcriptional profile of cells undergoing titanisation mirrors their cell-cycle phenotype. Consequently, the budding and mitosis genes, CDC11 and CLN1, were expressed early and peaked at 24hrs (a point at which budding is prevalent and most cells have a 1C DNA content), suggesting that the cells were predominantly in either G1 or M phase. CLN1 is required for releasing C. neoformans titan cells from G2 arrest (during cell cycle progression) [18] and therefore it is not surprising for that CLN1 is downregulated at day 3 (Fig 6B) when the budding index drops significantly (Fig 4C). However, it is intriguing that CLN1 is partially upregulated at day 5 and 7. In C. neoformans, CLN1 forms a critical balance between DNA replication and cell division [18]. Our data suggest that CLN1 is involved in the regulation of cell division during the first 24 hr of induction and in DNA replication during the unbudded phases at 5 and 7 days.

Although we did not study cell cycle regulation of C. neoformans titan cells, C. neoformans titans generated via our in vitro system differ from R265 titan cell by profusely budding after 3 days of induction (S7 Fig). C. neoformans titan cells can pass through the G2/M checkpoint (G2/M transition), commit to mitosis and produce buds [6,7,13,15]. Interestingly, the G2 arrested R265 titan cells actively proliferate when recultured in nutrient rich media, suggesting that they are not permanently growth arrested but rather in quiescence. In budding yeast, quienscent cells are known to be G1 arrested during stationary phase [42] whereas both G1 or G2 arrest is possible for Cryptococcus [41]. Therefore, it is not surprising that R265 quienscent titan cells show a G2 arrest. However, the seemingly high metabolic activity of these cells (when compared to log-phase R265 yeast cells) is intriguing and warrants further investigation in the future.

We also demonstrate that R265 titan cells produce daughter cells with a polyploid DNA content similar to their mother cells. In contrast, C. neoformans titan cells produce both haploid and aneuploid progeny [24], with sizes ranging between 5–7μm and 2–4μm respectively. Given that population heterogeneity in C. neoformans is associated with preferential dissemination to the CNS [43,44], it is possible that this lack of heterogeneity in C. gattii may contribute to the differences in disease etiology in this species. In particular, murine models have shown that C. neoformans isolates that produce high percentage of titan cells fail to disseminate to the brain and instead remain in the lung, consistent with pneumonia or chronic infection [13,24].

Through our in vitro model, we confirmed the requirement of host-relevant environmental cues (physiological temperature of 37°C and 5% CO2) for titan cell formation, as previously discovered [14,15]. We also highlighted cell density as a key regulatory factor for titan induction in C. gattii, as it is in C. neoformans. Consistent with previously reported in vitro induction model [14,15], production of R265 titan cells was inversely proportional to initial inoculum density such that no titan cells were produced at high density (106 cells/mL) and the maximum percentage was achievable at low density of 5x103 cells/mL. Interestingly, addition of the quorum-sensing peptide, Qsp1 [44,45] was able to significantly inhibit titan cell formation in our assay.

The fact that RPMI, but not the very similar cell culture medium DMEM, induced R265 titan cells enabled us to identify p-Aminobenzoic acid (pABA) as a major driver of titanisation. Interestingly, the titan induction effect of pABA was recapitulated when the C. neoformans strain, H99 was tested, although the very low titan formation capacity of this strain means that the effect was very weak (S1E Fig). The mechanism by which pABA triggers titanisation remains unclear at present. However, we note that pABA is an antifungal metabolite that has efficacy against several fungal plant pathogens such Fusarium graminearum, Magnaporthe oryzae, Rhizoctonia solani, Sclerotinia sclerotiorum and Valsa ambiens var. pyri. [46]. Since titanisation is linked to the fungal stress response, it may be that low dose pABA induces a mild stress that triggers titanisation. In this context modulation of the cell cycle and morphogenesis of Colletotrichum fructicola (a plant fungal pathogen) by pABA has been documented [47]. Alternatively, pABA’s well documented role in oxidative damage tolerance and the role of reactive oxygen species in titan cell induction [22,47,28] may suggest a role for reactive oxygen balance in this phenomenon.

Finally, using our in vitro protocol, we evaluated the capacity for titan cell formation between and within cryptococcal species. We found titanisation was particularly abundant within C. gattii/VGII (C. deuterogattii). Interestingly, Fernandes and colleagues reported a similar cell enlargement phenotype while screening for clinically relevant attributes in C. gattii [20]. To start to dissect the genetic regulation of this process, we tested and analysed the titanisation profile of a collection of parent/progeny crosses that we generated in our previous study [33]. It was striking to note the variation in this phenotype within recombinant progeny and, in particular, the very high rates of titanisation found in the offspring of ‘outgroup’ hybrids. Most of the progeny from this cross showed titanisation rates equal to or greater than that of R265 (the high titan cells generating parent). It is possible that this reflects a ‘hybrid vigour’ effect, resulting from the outcross. In the future, more detailed genomic investigation of these and other crosses may potentially facilitate a more comprehensive understanding of titan cell formation in this genus.

Overall, our titan induction protocol is an efficient and high throughput approach for producing titan cells at scale. By employing our in vitro protocol, we have discovered novel aspects of titanisation in C. gattii and revealed the separation of DNA replication and cell size increase. Together, we hope that this approach will provide a platform for the future mechanistic investigation of titanisation in this important group of pathogens.

Methods

Cryptococcal strains and culture conditions

Cryptococcal strains used in this study are listed in Table 2. Prior to use, cryptococcal strains were maintained on Yeast Peptone Dextrose (YPD) (1% yeast extract, 2% bacto-peptone, 2% glucose, 2% bactor-agar) agar at 4°C from which overnight cultures were prepared in YPD broth at 25°C, 200rpm.

Table 2

Cryptococcal species and strains used in this study.

Species and strains	Serotype	Genotype
C. gattii WM265	B	VGI	Clinical isolate, Brazil
C. gattii WM179	B	VGI	Clinical isolate, Australia
CBS8755		VGI
C384		VGI
NIH312	C	VGI	Clinical
B4546	C	VGI	Clinical
EJB11	B	VGI
MMCO8-897	B	VGI
CBS1508	B	VGI
CBS1873	B	VGI
C. gattii R265	B	VGIIa	Clinical isolate, Vancouver, Canada
C. gattii CDCR271	B	VGIIa	Clinical isolate, immunocompetent patient, Kelowna, British Columbia, Canada
C. gattii ENV152	B	VGIIa	Environmental isolate, Alder tree, Vancouver Island, Canada
C. gattii ICB180	B	VGII	Environmental isolate, Eucalyptus tree, Brazil
C. gattii CBS10089	B	VGII	Clinical isolate, Brazil
C. gattii CDC272	B	VGII	Clinical isolate, Greece
C. gattii B7735	B	VGIIb	Clinical isolate, Vancouver, Canada
C. gattii EJB11	B	VGIIb
C. gattii EJB52	B	VGIIc	Clinical isolate, Oregon, USA
C. gattii CBS6955	B	VGIII	Clinical isolate, Oregon, USA
CBS6993	C	VGIII	Clinical isolate, USA
CBS1622	B	VGIII
WM1243	B	VGIII
B13C		VGIII	Clinical isolate, Asia
CA2350		VGIII	Clinical isolate
CA1227		VGIII	Clinical isolate
C. gattii LA584	B	VGII	Clinical isolate, Colombia
C. gattii B5464	C	VGIII	Clinical isolate, USA
C. gattii WM779	C	VGIV	Clinical isolate, USA
B5742	B	VGIV
B5748	B	VGIV
CBS10101	C	VGIV
C. gattii CBS1010	C	VGIV	Veterinary, South Africa
C. neoformans H99	B	VNI	Clinical isolate, USA
C. neoformans Zc1	A	VNI
C. neoformans Zc8	A	VNI	Clinical, Zambia
C. neoformans Z12	A	VNI	Clinical, Zambia
C. neoformans 125.91	A	VNI	Clinical, Tanzania
C. neoformans Tu369-2	A	VNI	Environmental isolate, Mopane tree bark, Botswana
C. neoformans HAMDANC 3–1	A	VNII	Pigeon droppings, Belo Horizonte, Brazil
C. neoformans B3501	D	VNIV	Clinical isolate, USA
C. neoformans CBS6995	D	VNIV	Clinical isolate, USA
C. neoformans CBS8336	A	VNI	Wood of Cassia tree, Brazil

In vitro induction of Titan cells

Yeast cells from overnight cultures were collected (by centrifugation at 4000 r.p.m for 2 mins), washed three times with phosphate buffered saline (PBS), re-suspended in 3mL PBS and counted on a haemocytometer to determine cell densities. Except where otherwise noted, titan induction was achieved by inoculating 5x103 yeast cells in 1 mL serum free-RPMI 1640 within a 24 well tissue culture plate for 24 hours to 21 days at 37°C in 5% CO2 without shaking.

For generation of daughter cells, the 7 day old titan-inducing culture was passed through a >20μm cell strainer and >20μm-sized Titan cells were re-cultured in YPD overnight. Then, the daughter cells were isolated by filtering the overnight culture using a 15μm cell strainer and collecting the flow-through.

Cell size measurement

Cells recovered from titan induced or YPD grown cultures were washed in PBS and fixed with 50% methanol. After Indian ink staining for capsule visualization, cellular images were obtained using a Nikon TiE microscope equipped with phase-contrast 20X optics. Cell body and capsule sizes for individual cells were measured by using ImageJ software in combination with automated measurement based on Circle Hough Transformation algorithm [49-51].

Cell wall and capsule

Cells were fixed with 4% methanol-free paraformaldehyde for 10 mins and stained with calcofluor white (CFW, 10 μg/ml) for another 10 mins [13]. Total chitin was determined by flow cytometry on an Attune NXT instrument, with quantification of CFW staining using FlowJo software. For capsule visualization, cells were counterstained with India Ink (Remel; RMLR21518) and images acquired using a Nikon TE2000 microscope and analysed using ImageJ software.

The quorum sensing Qsp1 (NFGAPGGAYPWG) and scrambled Qsp1 [(AWAGYFPGPNG), control] peptides were synthesized at the University of Birmingham and then dissolved in steriled distilled water at 1mM and frozen at -20°C for future use. The peptides were then added to RPMI media at concentration of 20μM and R265 yeast cells from overnight culture were grown in the mixture at 37°C in 5% CO2 for 72 hr. Finally, the cell body size of R265 cells in the test media were analysed.

DNA content measurement Ploidy

RPMI and YPD grown cells were recovered, washed 3x in PBS, fixed in 50% methanol and stained with 3 ug/ml DAPI at 105 cells/mL. For each sample, about 10000 cells were acquired on an Attune NXT flow cytometer and the result was analysed using FlowJo v. 10.7.1. Cells were sorted for doublet and clump exclusion by using FSC-A vs FSC-H gating strategy (S3E Fig) and compared to control, YPD grown, yeast cells.

Identification of RPMI-titan inducing compound(s)/factors

RPMI-specific compounds (which are absent in DMEM) were supplemented to DMEM media and tested for capacity to induce titan cells in R265 (C. gattii). Accordingly, DMEM media was first supplemented with ‘RPMI-specific’ amino acids at their original concentration in RPMI: L-Glutamic acid (20 mg/L), L-Aspartic acid (20 mg/L), L-Arginine (200 mg/L), L-Glutathione (1 mg/L), L-Asparagine (50 mg/L) and L-Proline (20 mg/L). All the amino acids were purchase in powder form from Sigma-Aldrich and stock solutions were made by dissolving them according to manufacturer’s instruction.

To investigate if the glucose concentration difference between RPMI (high concentration) and DMEM (low concentration) was responsible for titan cell formation, DMEM was supplemented with D glucose (Purchase from Sigma-Aldrich) at a final concentration of 2000 mg/L (RPMI concentration). D glucose was purchased in powder form and stock solution was prepare according to manufacturer’s instructions.

Finally, DMEM media was first supplemented with other ‘RPMI-specific’ compounds at their original concentration in RPMI: Vitamin B12 (0.005 mg/L), Biotin (0.2 mg/L), and para-aminobenzoic acid [(pABA) 1.0 mg/L]. All these compounds were purchased from Sigma-Alrich in powder form and stock solutions were prepared according to manufacturers instructions.

Metabolic activity of titan

The metabolic state of R265 titan cells was confirmed by using FUN-1 reporter dye. The freshly obtained RPMI-induced 7 day old titan and YPD overnight (control) cells cultures were collected and aliquoted into two. Cells from an aliquot from the two conditions were heat-killed for 1 hr in a heating block (Stuart Heating Block). Both heat-inactivated and fresh cell samples were stained with Fun-l at 5μM for 30 mins. Microscopy images were taken with Zeiss Axio Observer at 63X magnification with fluorescent emission wavelength set at 590 nm.

RNA extraction and purification

Total RNA extraction was performed on uninduced (YPD grown) and titan-induced R265 cells by employing the protocol of QIAGEN (RNeasy Micro Kit [45]); Cat. No./ID74004) with slight modification. Samples of overnight YPD grown, R265 cultures and titan-induced cells of the different time-points (24 hr, 72 hr, 5 days and 7 days) were harvested, washed three times in PBS, adjusted to ~106 cells/mL and pelleted in 1.5mL Eppendorf tubes. The cell pellets were flash-frozen in liquid nitrogen and stored at -80°C overnight. The cells were lysed by mixing in 400μL of RLT buffer, transferring to a 2 mL lysing tubes (MP Biomedicals 116960100) and beating with a bead beater (MP Biomedicals 116004500 FastPrep 24 Instrument Homogenizer) for thorough cell disruption. The homogenized sample was centrifuged for 3 min at 10,000 xg at room temperature. The aqueous portion was separated, mixed with 70% ethanol at 1:1 ratio and transferred to an RNeasy Mini Spin Column. Finally, RNA extraction and purification were carried out as described in manufacturer’s protocol, including purification through a gDNA eliminator column to remove DNA contamination.

RT-qPCR and gene expression analysis

RNA was extracted from YPD grown and titan-induced samples and reversed transcribed (RT) to cDNA by using FastSCRIPT cDNA Synthesis protocol (Catalogue Number: 31-5300-0025R]. In brief, 15μL of RNA samples were mixed with 1μL of RTase and 4μL of FastSCRIPT cDNA Synthesis Mix (5X) before 30 min incubation at 42°C and subsequent 10 min incubation at 85°C. Quantitative PCR for the selected putative cell cycle genes was determined for each RT samples by mixing 2μL of the RT samples with 38μL master mixed of KAPA enzyme (KABA SYBR FAST qPCR Kits) and designed primers (S1 Table) and run in a real-time PCR detection system (CFX96 Touch Real-Time PCR Detection System; Ref. no.: 1845096). Gene expression level was obtained and normalized according to change difference with the housekeeping gene, GAPDH. Finally, the relative expression profile was expressed as a function of comparative threshold cycle (CT) by using the follow formula (III):

Delta CT = CT gene—CT GAPDH

Delta delta CT = CTgene-average Delta CT

Relative gene expression = 2-delta deltaCt

Statistical analysis

All analyses were performed with GraphPad Prism 6. The Shapiro-Wilk test was used to check normality of the data and either parametric (ANOVA) or non-parametric (Kruskal-Wallis or Mann Whitney, as described) tests used accordingly. Size distribution plots are shown as scatter plots with the mean highlighted and error bars reflecting the standard deviation.

Phenotypic analysis and impact of exogenous factors on titan cell induction in C. gattii.

A) Cell body size of R265 yeast cells before (in YPD) and after incubation in sterile RPMI and serum supplemented RPMI. The cells were grown in YPD overnight and in RPMI (sterile and serum-contained) for 3 days in 5% CO2 at 37°C and recovered for cell body size measurement. B) Micrograph of R265 yeast cells after grown in YPD for 7 days in 5%CO2 at 37°C. Scale bar = 15μm. C) pH of YPD amd RPMI before and during titan induction (24 hr and 7 days). Titan cell induction was performed with the R265 (C. gattii) and pH was measured. D) Effect of glucose on titan cell formation. DMEM media was supplemented with D glucose reaching 2000mg/L (the concentration in RPMI) and tested for capacity to induce cell enlargement (>15μm) as compared to RPMI. Titan induction was performed by incubating R265 yeast cells in the different media conditions for 24 hrs at 37°C in 5% CO2. E) The influence of pABA on titan induction in C. neoformans (H99). Cell body size was measured after H99 yeast cells were grown in the different induction media at 37°C in 5% CO2 for 72 hr.

(DOCX)

Click here for additional data file.

Effect of CO2 and reculturing on titan phenotypes.

A) Effect of 5% CO2 on cryptococcal growth. Cells incubated at 37°C in serum-free RPMI in 5% CO2 or atmospheric conditions for 24 hrs were assessed for colony forming unit (CFU) on YPD after to evaluate viability. Statistical significance was confirmed by a Two-tailed t-test where ** = p<0.05. B) Proliferation of R265 Titan cells after re-culturing in RPMI. Titan cells obtained from induced cultures after 7 days (A) were re-cultured in fresh, serum-free RPMI at 37°C in 5% CO2 for 24 hrs (B) and analysed microscopically for ability to bud. Scale bar = 15μm.

(DOCX)

Click here for additional data file.

Characterization of budding nature and titanisation of R265 titan-derived daughter cells.

A) Images showing the budding nature of daughter cells before and after titan induction for 24hr, 72 hr and 7 days. Scale bar = 5μm. B) Budding index of daughter cells before and after titanisation. C) Cell body diameter was measured microscopically, and percentage of titan cells determined based on >10μm cell size. The data represents three independent biological repeats and significance was confirmed by one-way ANOVA where, **** = p<0.0001 D) Daughter cells of R265 titan cells were isolated and returned to titan inducing condition and their ploidy was determined by DNA content measurement via flow cytometry before titan induction (green), after 24 hr (blue) and 7 days (brown) relative to R265 haploid yeast (red). E) Flow cytometry data showing the gating strategy employed to confirm cell ploidy of R265 titan daughter cells as compared to YPD grown yeast and (>20μm) filtered titan cells.

(DOCX)

Click here for additional data file.

Cell body diameter of 42 YPD grown and titan-induced cryptococcal isolates representing the different genotypes within the C. neoformans/gattii species complex.

All strains were induced for titan cell formation according to our in vitro protocol. The cell body diameter and ploidy (see S5 Fig) were determined after 72hrs as described in the Methods section. A, B, C, D) Cell body size distribution of isolates from C. gattii complex (VGI-VGIV) before induction (YPD) and after 72 hr of induction (RPMI). E, F) Cell body size distribution of isolates from C. neoformans (VNI-VNII) and C. deneoformans (VNIV) before (YPD) and after 72 hr induction (RPMI) respectively.

(DOCX)

Click here for additional data file.

DNA content of 42 YPD grown (red) and titan-induced (blue) cryptococcal isolates representing the different genotypes within the C. neoformans/gattii complex.

All isolates were induced for titanisation according our in vitro induction model (as mentioned in the Methods sections) and DNA content was assessed by DAPI staining and flow cytometry analysis.

(DOCX)

Click here for additional data file.

Ploidy of parent and progeny C. gattii strains before and after titan induction.

A) DNA content of YPD grown (red) and titan-induced (blue) of ingroup crossing [VGII(R265) x VGII (LA584)] strains and their 13 progeny (Alg23-Alg35) after 3 days. All isolates were induced for titanisation according to our in vitro induction model (as mentioned in the Methods sections) after DNA content was confirmed by DAPI staining and flow cytometry analysis. B) DNA content of YPD grown (red) and titan-induced (blue) of ingroup crossing [VGII(R265) x VGIII (B4564) strains and their 18 progeny (P1-P18) after 3 days. All isolates were induced for titanisation according to our in vitro induction model (as mentioned in the Methods sections) after DNA content was confirmed by DAPI staining and flow cytometry analysis.

(DOCX)

Click here for additional data file.

Morphology of C. neoformans cells before and after titan-induction via our in vitro model.

Microscopy images of YPD grown (A) and 72 hr titan-induced C. neoformans cells (B). Scale bar = 5μm.

(DOCX)

Click here for additional data file.

List of cell cycle phenotypes of titan induced and associated genes.

(DOCX)

Click here for additional data file.

Example timelapse movie showing titan cell formation in C. gattii R265 under in vitro conditions.

(AVI)

Click here for additional data file.

24 Feb 2022

Dear Prof May,

Thank you very much for submitting your manuscript "An in vitro method for inducing titan cells reveals novel features of yeast-to-titan switching in the human fungal pathogen Cryptococcus gattii" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

While all of the reviewers felt that much of the data presented in the manuscript was sound, there were significant concerns raised regarding the novelty of the findings. Specifically, the production of titan cells by C. gattii has already been demonstrated and it was felt that the authors did not provide sufficient comparison to C. neoformans titan cell formation to show that the differences in induction of titan cells they observed with C. gattii would not also occur with C. neoformans. As one example, without assessing the role of p-aminobenzoic acid in C. neoformans titan cell production it is difficult to determine the novelty of this finding to C. gattii. In addition, the data relating to the metabolic state of the cells, and whether the cells analyzed were in fact dead, raised additional questions about the novelty of these findings. While there were novel aspects of the study, such as the genetic crosses, it was felt that overall these were minimal in nature and thus did not raise the level of significance of the study to a level appropriate for publication in PLoS Pathogens. Some of the reviewers felt that the manuscript was interesting, and that appropriate inclusion of additional data would strengthen the significance and novelty of the observations to the broader PLoS Pathogens audience. It should be noted that this would require major revision of the manuscript and inclusion of additional experimental data.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Kirsten Nielsen, Ph.D

Guest Editor

PLOS Pathogens

Xiaorong Lin

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: THe authors were interested in characterrizing Titan cell formation in C. gattii based on a simple standardizable protocol. They brought new data on the genetics of TC formation using progeny experiments.

Reviewer #2: This study demonstrates that in vitro induction of the crucial titan morphotype in Cryptococcus neoformans extends to C. gattii. While this conservation itself isn’t a major surprise, the authors then demonstrate that differences in when and how C. gattii enters the titan state compared to C. neoformans is very biologically interesting, including the decoupling of polyploid formation with cell growth and budding. In addition, the seemingly quiescent state of the C. gattii titans raise additional questions about whether these cells are metabolically active. Depending on the metabolic activity of these cells, titan formation in C. gattii could include a persister-like state that could have fascinating biological implications.

Reviewer #3: The present manuscript describes the formation of titan cells in the fungal Cryptococcus gattii. This process has already been extensively described in C. neoformans, in particular in 2018 when three different groups described different in vitro conditions to induce this process.

The main strenght of the article is the description of all the ploidy changes associated to titan cell formation in C. gattii, and in particular, the differences with C. neoformans.

While the present article describes a new method to induce this process, many of the factors described have already been characterized in C. neoformans (temperature, strain variation, CO2, cell density effect, among others). For this reason, the novelty of the manuscript is limited. It has been also shown that C. gattii, and in particular this strain (CBS10514, R265) can form titan cells in vitro (reference 14).

Most of the results, and in particular those related to the ploidy and cell cycle, have been performed using a C. gattii isolate (R265, CBS10514), which was originally isolated from the Vancouver outbreak. This strain has been characterized in detail, and it has been shown that it was the result of a very rare event of same sex mating in fungi (Nature volume 437, pages 1360–1364 (2005)). For this reason, I feel that it is too premature to extrapolate the mechanisms described to the whole Cryptococcus gattii species, and ploidy and cell cycle mechanims should be described in more strains from different genotypes.

In general, the experiments are well planned and perform, although I feel that new experiments to address the specific mechanism required for titanisation in C. gattii should be performed.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: Incubation in YPD at 37°C and 5% CO2 (24H and 7 days) as a control condition of titan cell formation would be a nice addition to the author’s experiments. Add to Fig 1, 3D, 6. Indeed YPD at 25° is very different conditions. It should be interesting to identify the most important factors associated with Titan cell formation in C. gattii (37 and 5% C02 vs. medium??)

Line 87-88: could you add details on the use of serum in the first induction medium as in cell culture medium, fetal calf serum is often added to help mammalian cells to grow.

Line 115: All in vitro protocols evidenced a large vacuole occupying the cytoplasmic space.

Line 129-130: Fig 1E: the 3 large cells are looking as dead cells. Indeed, the nice phenotype inclufing the large vacuole is not visible on this picture. These cells have been characterized in Dromer’s lab pmid: 25827423 and look similar.

Please use merge picture (DIC+DAPI) for DAPI stainings. This will better show that the DAPI staining does not stain a nucleus but rather the remaining retracted cytoplasm of this dead cell. This point is important, because to describe a population with 16C, the authors should be sure that the staining is done on a population harboring living cells in a good shape.

More pictures of 7 days incubated C. gattii using DIC should be provided. Fig 1A are only showing 24h-incubated cells

Fig 4E: The authors should be sure that no “contamination with titan cells” occurs in their setting to avoid detecting polyploid Titan cells within the population of daughter cells. Please analyze and provide histograms of all population (in SSC and FSC channels) to assess the absence of big cells from the daughter cells purification.

Line 238-241. It is not clear in the text which conditions have been used for RNASeq analysis. Between 24h and 7days or at 24h and 7d?

Figure 6 (impact cell density and temperature and CO2) should appear earlier in the results. As these conditions should be fixed rapidly to standardize experiments and obtained the optimal Titan cell proportion.

Line 323: should be 6F instead of 6B

Fig 6G and 6H should be separated from the rest of Fig 6.

Fig 6H. why using 3 h rather than 7 days as the best time point to obtain a large number of TC.

The authors should investigate the pH of the different media tested. pH has an impact on Titan cell formation. Fig 6H that authors should investigate why pABA acts as an inducer of TC. The authors could add pABA in YPD in addition as another control to validate the impact of pABA on C. gattii TC formation.

As the authors tend to show that VGII produce more TC than the other lineages, they should include more strains to validate this observation on more than 9 cells. They also should include at least the same number of strains from the other lineages (only 2 strains for VGI or VGIII, VGIV). Indeed, VGIII VGIV or VGI are producing TC in the same range than that of VGII strains. May be just a matter of number of strains characterized.

Reviewer #2: Overall, I think that this manuscript is scientifically sound and interesting. Some additional contrasts to C. neoformans (see below) would increase the broad appeal of this paper. This manuscript also raises the following points:

Major points:

Lines 89, 96: The use of the term “giant” cells here is unclear. Since one of the first two groups to identify titan cells referred to them as “giant” cells, this could be interpreted as another term for titans rather than large cells that are not yet conclusively titan.

The data presented in figure 2 demonstrating that titan cells increase capsule size but do not appear to be actively growing is fascinating. Are these cells metabolically active? Could they represent a fungal phenomenon analogous to bacterial persister cells? Do titans have a long chronological lifespan compared to cells grown long-term in RPMI?

Does pABA induce titanisation in C. neoformans? Does iron block titanisation in C. neoformans?

Reviewer #3: - The ploidy study should be confirmed in more strains, not only R265.

- The role of p-aminobenzoic acid should be further explored. They should validate some of the hypothesis from the discussion. Does p-aminobenzoic acid have any effect on other strains and species, such as C. neoformans?

- It is not clear how this protocol compares to those described in 2018. Also, I think that the authors should take advantage of this protocol to provide insights on the molecular mechanisms that differentiate titan cell formation in C. gattii compared to C. neoformans. To increase the significance of the article, I would strongly recommend to perform an RNAseq experiment and look at global changes in gene transcription.

- Line 84, results. The authors state that the discovery that C. gattii forms titan cells in RPMI was observed during experiments in which the yeasts were cocultivated with alveolar macrophages. Was serum present in the initial experiment? It is surprising that the authors do not evaluate the effect of serum in their experiments, since this was one of the main inducing factors that induce in vitro titanisation in C. neoformans in two of the protocols described in 2018.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Line 30-31 : The critical role of titan cells for Cryptococcus neoformans to establish infection should be tempered.

Line 33. The discovery of this protocol is probably less serendipitous than the first observations in mice and for C. neoformans in vitro protocols.

Reviewer #2: Minor points:

The bars representing the median (? mean? not stated; should be) are very difficult to see in Figure 6H.

Reviewer #3: - Introduction should be rewritten. In particular, the authors should give more background, and summarize the findings of the article.

- The authors should revise the English style, and make sure that past tense is always used when explaning the results. Furthermore, they should avoid the use of "giant" cells, since it can be confused with "giant" macrophages.

- Statistics. The authors should include a specific statistics section in M&M. Furthermore, they have used ANOVA throughout the manuscript. However, this test can only be used when there is a normal gaussian distribution. And it seems that some of the size distribution are not normally distributed. They should assess the normality of all the samples (Shapiro-Wilk or Kolmogorov-Smirnov tests, and then evaluate whether a parametric (ANOVA) or non-parametric (i.e. Kruskal-Wallis) test should be applied.

- The definition of titan cell and thresholds used is confusing. In some cases, they use a threshold of 10 um, and in other figure they use a threshold of 15 um. This should be unified to 10 um and the data recalculated in all the figures (i.e, figure 6). If the threshold defined by the authors for titan cells is 10 um (cell body diameter), why do they use filters of 15 um pore size?

- It would be interesting to know if progeny from titan cells have a different chitin content compared to regular cells.

- I think that it is more correct to show the cell cycle and ploidy data (DAPI histograms) in lineal axis and not logarithmics, since the increse in DNA content is lineal.

- Figure 6. I would argue against the concept that C. gattii produces large haploid but non-titan cells. Titan cells should be defined by their size, since it is possible to obtain titan cells in different ways and through different mechanisms. I feel that from an imunological point of view, the main factor that determines immunological evasion is the size and not DNA content.

- Figure 6G. The authors should show the size of cells grown in YPD too.

- Regarding cell density, it has been shown in C. neoformans that cell density and quorum sensing phenotypes depend on a short peptide (QSP1), and that this peptide inhibits in vitro titanisation in C. neoformans (reference 14 and 15). I think that the authors should investigate if this peptide or other QS molecules have a similar effect in TC formation in C. gattii.

- Presentation of figure 5 is somehow confusing. I believe that it would be better to normalize the expression level in YPD to an arbitrary value of 1, and then present the rest of the data as fold-change compared to this condition.

- Table 2. Cryptococcus neoformans H99 strain is serotype A, VNI. This should be corrected.

- Line 576. The authors state in the RNA isolation section that it was done in 400 uL of "RNAase". Do they mean "RNAeay buffer"?

- RT-qPCR experiments. It seems that the samples were not treated with DNAse to ensure that there is not interference in the RT_qPCR of contaminant DNA. If this step was omitted, they authors should have carried out a control sample in which the RNAs are equally treated, but without the addition of reverse transcriptase, to make sure that these samples are negative. I recommend to confirm this, since this step can be very tricky, and DNA contamination can occur.

- Figure 4. It would be interesting to know what happens when titan cells recovered by filtration are incubated in fresh YPD and RPMI medium. Most probably, these cells are nutrient-depleted by day 2 of incubation, so it raises the issue of how do they behave when they are incubated again in a inducing and non-inducing medium.

- RPMI vs DMEM experiments. The authors should give the exact reference of the media used, and in particular, which glucose concentration do they contain. Furthermore, the authors should ensure that during the whole experiments (even through day 7), there is not a change in pH, and that these two media have the same buffering strenght for C. gattii. Yeast can significantly alter the pH medium by the secretion of protons or ammonia, and I am not sure how these media can buffer in these conditions without the addition of an additional buffer.

- Lines 358-363. The authors demonstrate that addition of iron inhibit titan cell development in C. gattii. This was also shown in C. neoformans (reference 14), so this should be highlighted.

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Alexandre Alanio

Reviewer #2: No

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at .

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here on PLOS Biology: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

17 Jun 2022

Submitted filename: Point by Point Response.docx

Click here for additional data file.

7 Jul 2022

Dear Prof May,

We are pleased to inform you that your manuscript 'An in vitro method for inducing titan cells reveals novel features of yeast-to-titan switching in the human fungal pathogen Cryptococcus gattii' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Kirsten Nielsen, Ph.D

Guest Editor

PLOS Pathogens

Xiaorong Lin

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: (No Response)

Reviewer #2: I think that the authors did an excellent job of addressing reviewer comments. The new data, particularly the revised figure 5, satisfied my concerns and make a good case for their scientific arguments.

Reviewer #3: The authors describe in this work in vitro conditions that efficiently induce the formation of titan-like cells in C. gattii. In the revised version, the authors have addressed most of the initial comments. I still believe that the main weakness is the lack of novelty because many of the factors described have already been identify in the literature as regulators of titan cells in C. neoformans. But even with this limitation, the authors provide a huge amount of information about titanization, and I believe that the article is significantly improved

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: none

Reviewer #3: Most of them have been addressed in the revision

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: none

Reviewer #3: Nothing to note

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Alexandre Alanio

Reviewer #2: Yes: Jessica Brown

Reviewer #3: No

12 Aug 2022

Dear Prof May,

We are delighted to inform you that your manuscript, "An in vitro method for inducing titan cells reveals novel features of yeast-to-titan switching in the human fungal pathogen Cryptococcus gattii," has been formally accepted for publication in PLOS Pathogens.

We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064