The Link between Morphotype Transition and Virulence in Cryptococcus neoformans

1Adaptation to the host environment by many eukaryotic pathogens is often companied by transition in cellular morphology,,,,,,,,. The ubiquitous fungal pathogen Cryptococcus neoformans causes more than half a million deaths each year. It can grow in the yeast form as well as the filamentous form. Earlier pre-genetic studies indicate an inverse relationship between filamentation and virulence,,,,,,. These studies also point to the potential of filament-specific antigens as vaccines against Cryptococcus infections,,.

2Because Cryptococcus typically grows in the yeast form and the morphological transition from the yeast form to the filamentous form appears to be coupled with mating, signaling pathways that lead to bisexual mating (a-α mating) and unisexual mating (mostly α-α mating) have been intensively investigated,,,. The roles of these signaling components in fungal pathogenicity are also scrutinized in animal models. However, accumulating evidence indicates that key signaling components that specifically lead to mating, such as those in the pheromone sensing pathway, have no or minimal direct effect on virulence,,,. Furthermore, conditions relevant to host physiology (e.g. aqueous environment, high temperatures, and high levels of CO2) are mating-suppressive, suggesting sex-independent mechanisms in orchestrating morphotype and virulence in Cryptococcus. Therefore, the existence and the nature of the link between morphological transition and virulence in Cryptococcus remain enigmatic.

3Although Cryptococcus morphological transition from the yeast form to the filamentous form is historically associated with mating, the observations that filamentation can be achieved in strains in the absence of key pheromone signaling components or meiotic genes,,,, lead us to hypothesize that pheromone signaling pathways are not essential or sufficient for filamentation per se, but they are critical in stimulating filamentation in response to mating cues. To test this hypothesis, we decided to examine the effect of constitutive activation of the pheromone signaling circuit on morphogenesis under mating-inducing and mating-suppressing conditions.

4It is known that the expression of genes in the pheromone signaling pathway, such as those encoding the pheromone Mf1α, the pheromone receptor Ste3α, the pheromone transporter Ste6, and the key pheromone response regulator Mat2 (Figure 1A), is low under mating-suppressing conditions but is dramatically higher during a-α bisexual mating (Figure 1B and data not shown),. We found that the expression level of these pheromone signaling genes in wildtype α strain H99 alone was low when cells were cultured on either mating-inducing condition (V8 agar) or mating-suppressing conditions (YPD agar and serum) (Figure 1B, C, D, E and F). This is consistent with the well-noted poor ability of the H99 strain to undergo unisexual mating. In fact, filamentation has never been observed when H99 was cultured alone under mating-inducing conditions (Figure 2A). We chose this strain to study the link between morphogenesis and virulence because H99 is one of the most virulent clinical strains tested in various animal models and it is also widely used as a reference strain in Cryptococcus research.

5When we placed the MAT2 gene under the control of the constitutively active promoter of GPD1 (glycerol-3-phosphate dehydrogenase 1) and introduced this construct to H99, the transcript level of MAT2 was dramatically increased under mating-inducing as well as mating-suppressing conditions (Figure 1C). As expected for a key regulator of the pheromone signaling, overexpression of MAT2 led to high expression of MF1α, STE3α, and STE6 under both mating-inducing and mating-suppressing conditions (Figure 1D, E and F). This result indicates that constitutively overexpression of MAT2 is sufficient to induce pheromone signaling circuit independent of mating cues.

6We next tested the effect of activation of pheromone signaling on filamentation under different conditions. The PGPD1-MAT2 conferred filamentation to H99 during unisexual mating (α cells alone) and it significantly enhanced filamentation during bisexual mating (a-α coculture) under the mating-inducing condition (Figure 2A). However, under mating-suppressing conditions, overexpression of MAT2 failed to stimulate filamentation in the α alone culture or in the a-α coculture (Figure 2A), and this was not due to insufficient activation of pheromone signaling. Effective activation of pheromone signaling in the PGPD1-MAT2 strain is supported by both the high expression levels of genes involved in pheromone signaling (Figure 1D, 1E, and 1F) and the formation of shmoo-like cells under both mating-inducing and mating-suppressing conditions (Figure 2B). Shmoo-like cells are typically observed when cells respond to mating signals prior to cell fusion. These observations indicate that activation of pheromone signaling alone is not sufficient to initiate filamentation under mating-suppressive conditions, including conditions relevant to host physiology. Thus mating signaling is unable to coordinate the yeast-filament morphological transition and virulence during infections.

7We previously showed that the deletion of ZNF2, which encodes a zinc-finger transcription factor, locked cells in the yeast form during mating without impairing pheromone signaling. This suggests that Znf2 is not essential for mating signal relay; rather, it is crucial for filamentation. Although Znf2 functions downstream of Mat2 during mating and its gene expression was significantly induced by MAT2 overexpression under the mating-inducing condition (Figure 2C and 2D), activation of the pheromone signaling pathway was unable to induce ZNF2 expression in the absence of mating stimuli. This was evidenced by the low expression level of ZNF2 in the PGPD1-MAT2 strain under mating-suppressing conditions (Figure 2C and 2D). The ability of Cryptococcus to undergo filamentation correlates with the expression level of ZNF2, but not that of MAT2 (Figure 1C, Figure 2A, 2C and 2D). Thus, Znf2 could be a master regulator that dictates Cryptococcus morphotype irrespective of environmental stimuli or mating type.

8To test this hypothesis, we constructed the PGPD1-ZNF2 strains. Indeed, the PGPD1-ZNF2 triggered filamentation in Cryptococcus strains of either mating types a or α in both serotype A and serotype D backgrounds under all tested conditions, including those that are inducing or suppressive to mating (Figure 3A and Figure S1). In contrast to the PGPD1-MAT2 strain, filaments produced by the PGPD1-ZNF2 strain under mating-inducing condition maintained their filamentous morphology after being transferred to mating-suppressive conditions (Figure S2). However, it is notable that the PGPD1-ZNF2 strain produces more robust hyphae under mating-inducing condition, suggesting that other factors induced under mating-inducing condition could further activate Znf2.

9The PGPD1-ZNF2 also conferred filamentation to mutants that harbor deletions in the key mating components under various conditions tested (Mfα1-3, Mat2, or Ste12 functioning in a branching pathway in pheromone signaling) (Figure 3B). To confirm that filamentation conferred by Znf2 activation is not due to some cryptic restoration of mating ability, we measured the efficiency of cell fusion of the wildtype, the mat2Δ mutant, and the mat2Δ+PGPD1-ZNF2 strain during bisexual a-α mating. Indeed, overexpression of ZNF2 did not rescue the cell fusion defects of the mat2Δ mutant (Figure 3C). Consistently, gene ontology analyses of our previous transcription data indicate that Znf2, unlike Mat2, does not regulate genes involved in the cell fusion event critical for mating (Figure S3). Taken together, the results indicate that filamentation can be independent of mating and Znf2 is one key determinant of this sex-independent morphogenesis.

10To verify the correlation of ZNF2 expression and Cryptococcus morphology, we constructed the ZNF2 gene driven by two inducible promoters: the galactose-inducible GAL10 promoter (data not shown) or the copper transporter CTR4 promoter (Figure 3D) (copper deprivation–on; copper repletion–off). Transformation of the PGAL10-ZNF2 or the PCTR4-2-ZNF2 construct into wildtype either the serotype D reference strain JEC21 or the serotype A reference strain H99 conferred filamentous growth under promoter-inducing conditions. These strains grew as yeasts under promoter-repressive conditions (Figure 3D and data not shown). Increasing the concentration of the copper chelator BCS (inducer) increased the frequency of filamentation in the PCTR4-2-ZNF2 strain (Figure 3D), indicating that the expression level of ZNF2 dictates Cryptococcus cellular morphology.

11To examine the effect of ZNF2 on the dynamic morphological transition, we incubated the PCTR4-2 -ZNF2 strain in H99 background in liquid YPD medium containing 200 µM BCS (inducer) and examined cell morphology over time. Morphological transition from the yeast form to the filamentous form completed by 60 hours (Figure 4). At this time, the hyphae were transferred to YPD medium containing copper sulfate (inhibitor). Cryptococcus cells then switched from the filamentous form to the yeast form over time (Figure 4). The control of bi-directional morphological transition by Znf2 is also observed when cells were cultured in serum (data not shown), indicating that this control is independent of environmental cues. These results demonstrate that (i) the expression level of ZNF2 determines Cryptococcus cell morphology: high expression level of ZNF2 drives the cells to the filamentous form and low expression level of ZNF2 renders cells unicellular yeast; (ii) Znf2 is necessary and sufficient to initiate morphological transition; (iii) High Znf2 activity is required to maintain cells in the filamentous morphotype.

12The relationship between morphotype and pathogenicity is typically defined through studying morphological mutants that are otherwise isogenic to the wildtype strains and are able to maintain given morphotype under host relevant conditions, even though mutants with such extreme phenotypes are unlikely to be encountered clinically due to natural selections in the host,,,. For Cryptococcus, host physiological environment (e.g. high body temperature, aqueous environment, and high levels of CO2) is extremely inhibitory to mating. Consistently, constitutively activated mating signaling induced filamentation under mating-inducing conditions, such filaments could not be maintained when transferred to in vitro conditions that mimicked host physiological environment (Figure S2). In contrast, the PGPD1-ZNF2 strain can readily initiate and maintain filamentous growth under such host-relevant conditions (Figure S2). Thus ZNF2 overexpression strains could serve as a model to investigate the relationship between morphotype and pathogenicity.

13We tested the virulence of the wildtype H99 and the PGPD1-ZNF2 strain in the murine inhalation model of cryptococcosis. The PGPD1-ZNF2 strain exhibited heterogeneity in cell morphology and a mixture of cell types is always present in this strain. To obtain accurate inoculation and to avoid potential problems caused by differences in cell types at initial infection, only cells in the yeast form were used for animal inoculation. Remarkably, the PGPD1-ZNF2 strain was completely avirulent (Figure 5A). By day 60 post infection (DPI 60) when the study was terminated, the PGPD1-ZNF2 cells were either completely cleared from animal lungs or existed in very low numbers (1000 fold lower than the original inocula). We further examined the fungal burden in the lungs and the brain of animals infected with H99, the znf2Δ mutant, and the PGPD1-ZNF2 strain at DPI 10 before any animal succumbed to cryptococcosis. Consistent with the animal survival rates, the lung fungal burden in animals infected with the znf2Δ mutant and the PGPD1-ZNF2 strain was 236% and 0.6% respectively compared to those infected with the wildtype (Figure 5B). The brain fungal burden showed a similar trend with larger variations due to individual differences in the timing of dissemination in this inhalation model (Figure S4), and no fungal cells were recovered from the brains of animals infected by the PGPD1-ZNF2 strain. To examine the effects of Znf2 on fungal morphology in vivo, we infected animals intranasally with H99, the znf2Δ mutant, and the PGPD1-ZNF2 strain and performed histological examination of lung tissues at DPI 1, 7, and 12. Remarkably, even though only yeast cells from the PGPD1-ZNF2 strain were used in the original inoculation into animals, lungs infected by the PGPD1-ZNF2 strain contained Cryptococcus cells of mixed morphology: yeast, pseudohyphae, and hyphae in all the time points examined (Figure 5C and Figure S5). This is consistent with the morphological heterogeneity of the PGPD1-ZNF2 strain in vitro (Figure S2). In comparison, only yeast cells were observed in the wildtype H99 or the znf2Δ mutant infected animals (Figure 5C and Figure S5). This histological examination indicates that activation of Znf2 can drive filamentation in vivo.

14Tolerance of host temperatures is a pre-requisite of fungal virulence. In some fungal pathogens, morphological changes are often a response to temperature and some morphological defective mutants lose the ability to cause diseases in mammalian hosts due to growth inhibition by high temperatures in vivo. To determine if alteration of virulence potential in the znf2 mutants are simply due to altered sensitivity to high temperature, we compared the growth of the wildtype H99, the znf2Δ mutant, and the PCTR4-2-ZNF2 strain at 30°C and 37°C on a variety of media via the spot assay. No apparent growth defects were observed in the znf2Δ mutant or the ZNF2 overexpression strain when compared to the wildtype under the conditions tested (Figure S6). Furthermore, the observation that the ZNF2 overexpression strain was capable of amplification during early stages of infection based on the fungal burden time course experiment (Figure S7) also suggests that factors other than growth inhibition by high temperature are mainly responsible for the effects of Znf2 on virulence.

15As morphological changes reflect changes in cell surface properties, we predict that Znf2 controls cell surface constitutes. One property likely regulated by Znf2 is cell adhesion, as supported by the following observations. First, increasing the ZNF2 expression led to increasingly wrinkled colony morphology and flocculation (Figure 3D, and Figure 6A, B and C). Both phenotypes are likely caused by increased expression of flocculins (adhesins or adhesion proteins), as previously shown in bacteria and in yeasts,. Second, aerial hyphae of the ZNF2 overexpression strains formed on solid media also tended to attach to each other, forming bundles (Figure 6D), as observed in flocculated strains of the filamentous fungus Ashbya gossypii. Third, deletion of ZNF2 impairs agar invasion whereas overexpression of ZNF2 remarkably promoted invasive growth (Figure 6E), and invasive growth reflects cell-substrate adhesion. The results suggest that Znf2 plays a pivotal role in morphogenesis-associated cell flocculation in Cryptococcus.

16Given that Cryptococcus strains with increased flocculation are reduced in virulence,, this transcription factor likely impacts pathogenicity at least partly through its effects on cell adhesion. Ontology analysis of our previous transcriptional profiling data revealed that of those genes that are differentially expressed in the znf2Δ mutants, 23% encode secretory proteins based on WoLF PSORT prediction (http://wolfpsort.org/) (Figure 7A). We selected 9 such genes and examined their transcript level in a ZNF2 overexpression strain incubated in serum at 37°C in 5% CO2 by quantitative realtime PCR. All genes tested were also differentially expressed in the ZNF2 overexpression strain (Figure 7B).

17We overexpressed these 9 genes using the constitutively active GPD1 promoter and examined if their overexpression could recapitulate some of the phenotypes caused by the ZNF2 overexpression (Figure 7C). Interestingly, strains with overexpression of CNAG_00795 (designated as CFL1: Cell FLocculin 1) formed extremely wrinkled colonies, like ZNF2 overexpression strains (Figure 6A). Interestingly, the expression of CFL1 was also most dramatically induced by the ZNF2 overexpression (Figure 7B). Because acapsular Cryptococcus mutants also form wrinkled colony, we examined capsule production in the CFL1 overexpression strain and cfl1Δ mutants. No apparent defect in capsule production was detected based on microscopic examination (data not shown).

18To confirm that cell adhesion is indeed caused by increased CFL1 expression, we then constructed PCTR4-2-CFL1 strains. These strains grew as yeast cells in liquid cultures. A sharp increase in cell aggregation was observed when PCTR4-2-CFL1 cells were cultured under promoter-inducing conditions, a reminiscence of some of the phenotypes of the PCTR4-2-ZNF2 strains (Figure 7D).

19To further confirm that CFL1 is regulated by Znf2, we engineered a reporter strain where ZNF2 expression is inducible by galactose and the fluorescent Cfl1 is driven by its native promoter. We grew the reporter strain under mating-suppressing conditions to avoid complication due to potential activation of mating signaling. Under such conditions, the colony formed by the reporter strain became fluorescent and wrinkled when the ZNF2 expression was induced in the presence of galactose (Figure 8A and B), while the colony was non-fluorescent and smooth when the ZNF2 expression was inhibited in the presence of glucose (Figure 8A and B). Thus the expression of the fluorescent Cfl1 is driven by Znf2. Taken together, Znf2 triggers morphological switch as well as flocculation (cell adhesion), and its downstream factor Cfl1 regulates cell adhesion.

20We examined the sub-localization of Cfl1 using a strain harboring the mCherry fused Cfl1 protein driven by its native promoter. Because Cfl1 is induced during mating and controlled by key components of mating signaling (Figure S8A and B), we examined microscopically the expression of CFL1-m-cherry during mating. Cfl1 was rarely detected in yeast cells (Figure 8C), but it was highly expressed in hyphae during both unisexual mating and bisexual mating (Figure 8D). The fluorescent Cfl1 delineated the periphery of hyphal cells, consistent with the function of adhesins on the cell surface and the prediction that Cfl1 is a secretory protein based on the presence of an N-terminal signal peptide for secretion.

21Secretion is required for Cfl1's function as an adhesin. This is supported by the observation that overexpression of the fluorescent Cfl1 that lacks the N-terminal signal peptide [Cfl1(sigPΔ)-mCherry] failed to confer wrinkled colony morphology or cell aggregation to Cryptococcus (Figure S9 and Figure 8E). This is not due to a failure of producing the mutant allele protein, as abundant Cfl1(sigPΔ)-mCherry protein was produced by the cells (Figure 8E). However, no fluorescence was detected from the culture supernatant (Figure 8F), indicating defects in secretion. A few other fungal adhesins are also reported to be associated with cell surface as well as being released into surrounding environment,. Such property may facilitate their roles in mediating both cell-cell adhesion and cell-substrate adhesion, and it may also help circumvent the blockage by other extracellular components. Consistent with its role as an adhesin, Cfl1 regulates a broad spectrum of cell adhesion-related biological processes, including complex colony morphology, and formation of different biofilms (Figure S10).

22Remarkably, deletion of CFL1 dramatically reduced hyphal production during either bisexual or unisexual mating while overexpression of CFL1 enhanced the hyphal formation (Figure 9A and B). Thus, both the expression pattern of CFL1 and the observed effects of CFL1 deletion or overexpression on hyphal development indicate the importance of this adhesin in hyphal morphogenesis. Like the ZNF2 overexpression strain, hyphae formed by the PGPD1-CFL1 strain on YPD medium (mating-suppressive) tended to attach to each other, forming bundles (Figure 6C and Figure 9A).

23Previous studies implicate an inverse association between flocculation and virulence in Cryptococcus,. Consistently, we found that overexpression of CFL1 resulted in attenuation in virulence, indicating that Cfl1-mediated cell adhesion negatively modulates virulence (Figure 9C). Consistently, organ fungal burdens were maintained at low level in the PGPD1-CFL1 and PGPD1-ZNF2 infected animals at DPI 7, whereas the wildtype H99 strain proliferated significantly (Figure 9D). Unlike the PGPD1-ZNF2 strain, the PGPD1-CFL1 strain was not completely avirulent and the PGPD1-CFL1 strain proliferated significantly when examined at DPI 12 (Figure S11). This is surprising but not unexpected as the impact of ZNF2 overexpression is likely the combinational effect of additional adhesion proteins and morphogenesis factors. As noted for znf2 mutations, deletion or overexpression of CFL1 did not cause any apparent change in growth compared to wildtype when cultured at 37°C with 5% CO2 (Figure S12A and B). Cells aggregated when CFL1 was overexpressed at both 30°C and 37°C as expected.

24C. neoformans is the major fungal pathogen from the phylum Basidiomycota in the Kingdom Fungi. Its morphological differentiation is typically heterogeneous and stochastic, and has been historically associated with mating. Pheromone signaling is the master regulation system in fungal mating, and it is required for early mating events such as cell recognition, mating projection formation, and initiation of cell contact and cell fusion,,. However, increasing evidence implies that filamentation in Cryptococcus is a plastic process that is not limited to mating or the production of recombinant progeny: Filamentation is occasionally observed under mating-suppressing conditions, even in some attenuated strains isolated from infected host tissues,,,; Filamentation can occur in the absence of some key components of pheromone signaling or meiosis machinery,,,. Thus, filamentation could be used in behaviors unrelated with mating, such as foraging nutrients or defending predation. Such sex-independent cellular differentiation likely involves signaling pathways in response to cues other than the mating signal.

25Here we show that sex-independent morphogenesis is linked with virulence in this fungus. We further demonstrate that the transcription factor Znf2 plays a pivotal role in cryptococcal morphological transition, and it is necessary and sufficient to drive filamentation irrespective of environmental cues, mating types, or pheromone signaling. Znf2 not only controls morphogenesis in vivo, but also the ability of this fungus to cause diseases. Thus Znf2 provides the key link between morphogenesis and virulence in Cryptococcus.

26The exact mechanism by which Znf2 controls morphogenesis and links Cryptococcus pathogenicity is of great interest. Previous and this current in vitro studies indicate that Znf2 does not affect typical Cryptococcus virulence traits (e.g. melanization, capsule production, growth at high temperatures, growth in minimal media, and resistance to salt or H2O2, ). Although the PGPD1-ZNF2 strain is avirulent, this strain was capable of propagation during the first two weeks of infection (Figure S7). This is in contrast with other avirulent strains such as cna1 or capsule mutants, which are less fit under various stress conditions and are rapidly cleared by the host,. These lines of evidence point to new traits regulated by Znf2 that influence pathogenicity.

27Our observation that genes encoding secretory proteins are enriched within the regulon of Znf2 emphasizes the importance of changes in cell surface during morphogenesis. Given that Cryptococcus strains with increased flocculation have been noted to be reduced in virulence,, Znf2 likely impacts pathogenicity at least partly through its effects on cell adhesion (flocculation). Cell adhesion mediated by microbial pathogens usually involves a repertoire of extracellular adhesion proteins. One of Znf2's downstream factors, Cfl1, is a prominent adhesion protein which orchestrates filamentation, cell adhesion, and virulence. To our knowledge, Cfl1 is the first Cryptococcus adhesin discovered. Interestingly, Cfl1 does not resemble any known adhesins characterized in ascomycetous fungi in terms of primary sequences and functional domains based on Pfam prediction (http://pfam.sanger.ac.uk/). There are four other homologues of CFL1 in the genome of Cryptococcus and in some other species in the phylum of Basidiomycota (Figure S13), in which no adhesin has been identified so far. This suggests that Cfl1 and its homologues represent a novel adhesion family specific to Basidiomycota.

28Unlike Znf2, overexpression of CFL1 attenuates but does not abolish Cryptococcus virulence in the murine model of cryptococcosis. This is not unexpected as studies show that microbes are typically endowed with multiple adhesins. The master regulator Znf2 likely controls additional adhesins and other morphogenesis factors, and it is the orchestrated effects of its downstream targets that give rise to its overall impact on morphogenesis and virulence. Further characterization of Cfl1, other adhesins, and morphogens downstream of Znf2 can help parse out the effects of cell morphotype and other cell properties (e.g. changes in cell surface proteins like adhesins) on Cryptococcus virulence. Such investigation may lay a foundation for future endeavors to develop vaccines or alternative therapies against cryptococcosis.

29This study was performed according to the guidelines of NIH and Texas A&M University Institutional Animal Care and Use Committee (IACUC). The animal models and procedures used have been approved by the Institutional Animal Care and Use Committee (IACUC) at Texas A&M University (protocol number: 2011-22).

30Strains used in this study are listed in Table S1. For mating assays, parental strains (a and α) with equal number of cells were cocultured together on V8 medium in the dark at 22°C, and mating was examined microscopically for formation of mating hyphae and spores. For cell fusion assays, the coculture of marked parental strains were removed after 48 hours of incubation on V8 medium, washed, and plated on selective media to select fusion products at 37°C as described previously,,. For self-filamentation assays, cells were patched on V8 medium alone and hypha formation was examined microscopically. Phenotypical assays in vitro were performed as previously described. The serotype A strain H99 is highly virulent and has been widely used in pathogenesis studies. Thus strains generated in this genetic background were used in the animal experiments and many of the in vitro characterization experiments. However, because wildtype H99 has not been observed to undergo unisexual mating and its bisexual mating is rather weak compared to the well-characterized but less virulent serotype D strains such as JEC21 and XL280, strains generated in these genetic backgrounds were used in some of the morphogenesis and mating assays.

31Plasmids and primers used in this study are listed in Table S2 and S3. For gene deletion, overlap PCR products with an appropriate selection marker connected with the 5′ and 3′ flanking regions of gene of interests were introduced into Cryptococcus strains by biolistic transformation and transformants with homologous replacement were selected as described previously. For overexpression, genes were amplified by PCR and the amplified fragments were digested and inserted into pXL1 after the GPD1 promoter. The PGPD1 of the resulting plasmids was replaced with either the PCTR4-2 or the PGAL10 to generate the copper or the galactose inducible system. The PCTR4-2 and the PGAL10 were amplified from the plasmid pNAT/CTR4-2 and H99 genomic DNA respectively,.

32Because Cfl1 contains a predicted secretory signal peptide at its N-terminus, the mCherry was fused to the C-terminus. The fragment including CFL1 coding region and 1 kb upstream sequences (NCfl1) was pieced together with the mCherry by an overlap PCR. The resulting products were introduced into plasmid pXL1 to generate pXL1-NCfl1-mCherryA (for the serotype A H99 allele) and pXL1-NCfl1-mCherryD (for the serotype D JEC21 allele). The CFL1-mCherry without the CFL1 promoter was amplified and introduced into pXL1 to produce the plasmid pXL1-Cfl1-mCherry. The PGPD1 in pXL1-Cfl1-mCherry was replaced with the PCTR4-2 to generate plasmid pXC-Cfl1-mCherry. To construct overexpression of the fluorescent Cfl1 that lacks the N-terminal signal peptide [Cfl1(sigPΔ)-mCherry], primers primers Linlab948 and Linlab864 were used to generate CFL1(sigPΔ)-mCherry allele and pXC-Cfl1-mCherry was used as the template. The resulting PCR product was introduced into pXC to produce pXC- Cfl1(sigPΔ)-mCherry. Plasmids were linearized before introduced into relevant Cryptococcus strains. To examine the sub-cellular localization of Cfl1::mCherry, strains were grown on V8 agar medium at 22°C for 72 hrs before examined with a BX50 (Olympus) microscope.

33Total RNA was purified using the purelink RNA purification kit (Invitrogen) and was used as the template for the first strand cDNA synthesis using the Superscript III cDNA synthesis kit (Invitrogen). Relative expression level of selected genes was measured by real time PCR using power SYBR qPCR premix reagents (Invitrogen) in a Realplex system (Eppendorf). Primer efficiency was determined by serially diluting the cDNA and monitoring DNA amplification by real-time PCR. Primers for qPCR used in this study are listed in Table S3. Gene-expression levels were normalized using the endogenous control gene TEF1. The relative transcript levels were determined using the comparative CT method as described previously.

34RNA was separated on agarose gels blotted to nylon membrane. Redi-Prime II kit (Amersham) was used to generate probes. The C. neoformans actin gene transcript served as a control. mRNA purification was performed using the PolyATtract mRNA Isolation System III (Fisher) according to the manufacture's instruction.

35The cells were cultured in 96-well microtiter plates under a variety of growth conditions. The air-liquid interface biofilm was only observed in CFL1 overexpression strains. The strains were grown in YPD liquid medium for 8 days. Crystal violet method was used for the quantitative assessment of the ability of Cryptococcus strains to form biofilm as previously described.

36Animals were infected essentially as previously described,. Groups of 6- to 8-week-old female A/J mice (Jackson Labs) were infected intranasally with 1×105 Cryptococcus cells in 50 µl PBS. For the PGPD1-ZNF2 strain, the culture of cells with mixed morphotype was centrifuged briefly at a low speed to allow the enrichment of yeast cells on the top. The top culture was then centrifuged again and only yeast cells were collected for infection. Ten mice per group were used for survival studies, and four or five were used for organ fungal burden studies and histological examinations. For organ fungal burden studies, fungal CFUs from lungs, kidneys, spleen, and the brains of sacrificed mice at each time point were measured as described previously,. Dunnett's two-tailed t test was used to test statistical differences (P≤0.05). For histological examinations, organs from the sacrificed animals were fixed in 10% formalin, embedded in paraffin, sectioned at 5 µm in thickness, and stained with hematoxylin and eosin (H&E) and Gomori methenamine silver (GMS) as previously described,. For mortality studies, the infected animals were monitored until all mice were sacrificed due to sickness or up to DPI 60 when the experiment was terminated. If the experiment was terminated, surviving animals were examined for the presence of Cryptococcus cells. Statistical significance (P≤0.05) of the survival data between different groups was assessed by the Mantel-Cox log-rank test.

37C. neoformans var. grubii (H99): ZNF2 (CNAG_03366); MAT2 (CNAG_06203); STE3α (CNAG_06808); STE6 (CNAG_03600); MF1α (CNAG_07407), CFL1 (CNAG_00795) and other secretory protein encoding genes controlled by Znf2 (CNAG_00596, CNAG_00925, CNAG_01211, CNAG_05778, CNAG_07422, CNAG_06239, CNAG_06411, CNAG_05729), KEL1 (CNAG_01149), CDC10 (CNAG_01373), CDC12 (CNAG_01740), cnCDC11 (CNAG_02196), cnMUC1 (CNAG_03234), cnCDC24 (CNAG_04243), cnCDC3 (CNAG_05925).

38C. neoformans var. neoformans (JEC21): ZNF2 (CNG02160), MAT2 (CNM02020), STE12α (CND05810), CFL1 (CNA07720). (Gene ID numbers were obtained from either NCBI Entrez or the Cryptococcus genome website at the Broad Institute http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans/MultiHome.html)