# Role of Cln1 during melanization of Cryptococcus neoformans

Rocío García-Rodas, Nuria Trevijano-Contador, Elvira Román, Guilhem Janbon, Frédérique Moyrand, Jesús Pla, Arturo Casadevall, Oscar Zaragoza

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Rocío García-Rodas, Nuria Trevijano-Contador, Elvira Román, Guilhem Janbon, Frédérique Moyrand, et al.. Role of Cln1 during melanization of Cryptococcus neoformans. Frontiers in Microbiology, 2015, 6, pp.798. 10.3389/fmicb.2015.00798. pasteur-03940603

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1 Role of Cln1 during melanization of *Cryptococcus neoformans*

2 Rocío García-Rodas ¹&, Nuria Trevijano-Contador ¹, Elvira Román ², Guilhem

3 Janbon ³, Frédérique Moyrand ³, Jesús Pla², Arturo Casadevall ⁴ and Oscar

4 Zaragoza ¹*

5 ¹ Mycology Reference Laboratory, National Centre for Microbiology, Instituto de Salud

6 Carlos III, Majadahonda, Madrid, Spain

7 ² Department of Microbiology. Pharmacy Faculty. Complutense University of Madrid.

8 ³ Unité Biologie et Pathogénicité Fongiques, Institut Pasteur, Paris, France

9 ⁴ Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg

10 School of Public Health, Baltimore, MD, USA.

11

12

13 &Present address : Institute of Biology Valrose (iBV) UMR CNRS7277 –

14 INSERM1091 – University of Nice-Sophia Antipolis. Centre de Biochimie, 06108

15 Nice, FRANCE

16 *Corresponding Author. E-mail: ozaragoza@isciii.es

17

18 Running title: Cln1 regulates melanization in *Cryptococcus neoformans*

19 Key words: Melanin, cell wall, laccase, *Cryptococcus*, *CLN1*

20

21

22

23
24 Abstract

25 *Cryptococcus neoformans* is an opportunistic fungal pathogen that has several well-26 described virulence determinants. A polysaccharide capsule and the ability to produce 27 melanin are among the most important. Melanization occurs both *in vitro*, in the 28 presence of catecholamine and indole compounds, and *in vivo* during the infection.

29 Despite the importance of melanin production for cryptococcal virulence, the 30 components and mechanisms involved its synthesis have not been fully elucidated. In 31 this work, we describe the role of a G1/S cyclin (Cln1) in the melanization process.

32 Cln1 has evolved specifically with proteins present only in other basidiomycetes. We 33 found that Cln1 is required for the cell wall stability and production of melanin in C. 34 *neoformans*. Absence of melanization correlated with a defect in the expression of the 35 *LAC1* gene. The relation between cell cycle elements and melanization was confirmed 36 by the effect of drugs that cause cell cycle arrest at different phases, such as rapamycin 37 and benomyl. The *cln1* mutant was consistently more susceptible to oxidative damage 38 in a medium that induces melanization. Our results strongly suggest that a novel and 39 hitherto unrecognized role for *C. neoformans* Cln1 in the expression of virulence traits.

40

41

42
43 **Introduction**

44

Melanins are a dark, hydrophobic, negatively charged pigments that are widespread in nature (Nosanchuk and Casadevall, 2003). The structure of melanins remain unidentified, so they are defined as pigments that are complex polymers with a high molecular mass, amorphous nature, acid resistance and with a stable free radical signal (White, 1958; Jacobson, 2000; Wakamatsu and Ito, 2002). There are several types of melanins: eumelanins, pheomelanins, alomelanins and piomelanins (Carreira et al., 2001; Wakamatsu and Ito, 2002; Plonka and Grabacka, 2006). Some fungal melanins derive from the precursor molecule 1,8-dihydroxynaphthalene (DHN), such as those from *Wangiella dermatitidis* and *Alternaria alternata*, and are produced from endogenous substrates. Alternatetively, some fungi produce melanin from L-3,4 dihydroxyphenylalanine (L-DOPA) (Eisenman and Casadevall, 2012).

Melanins contribute to the virulence of pathogens and increase resistance to environmental damage as well (Rosa et al., 2010). In *C. neoformans*, genes involved in melanization contribute to host death and dissemination from lungs (Salas et al., 1996; Noverr et al., 2004). Melanization in *C. neoformans* is catalyzed by laccase, a cell wall associated diphenoloxidase that catalyses the oxidation of diphenolic compounds to their respective quinones (Chaskes and Tyndall, 1978). However, *C. neoformans* cannot use tyrosine as a substrate to produce melanin (Nurudeen and Ahearn, 1979). Two laccase encoding genes, *LAC1* and *LAC2*, have been identified in *C. neoformans*, with *LAC1* being the main producer of melanin (Pukkila-Worley et al., 2005). In addition, other genes including *VPH1*, *CCC2*, *ATX*, *CHS3*, *MBF1* and *XRN1* are also required for melanization, although in most cases their mode of action is not well
67 characterized (Erickson et al., 2001; Zhu et al., 2003; Walton et al., 2005; Wollschlaeger
68 et al., 2014).

69 We have been recently interested in the role of cell cycle elements in the regulation of
70 the expression of cryptococcal virulence factors, with special emphasis on the capsule.
71 During the course of the characterization of *cln1*, a cyclin mutant of *C. neoformans*
72 (Garcia-Rodas et al., 2014) we found out that it was unable to melanize. Cln1 is a G1
73 cyclin in *C. neoformans*. While it is not essential for cell cycle progression, it is
74 responsible for a significant delay in the transition between G1 and S phases (Garcia-
75 Rodas et al., 2014). Little is known about cell cycle progression in *C. neoformans*, but
76 during exponential phase, budding and DNA synthesis occur simultaneously (Berman,
77 2006). However, at the end of exponential phase, budding is delayed and cells are
78 arrested at G1 or G2 phases (Takeo et al., 1995; Virtudazo et al., 2010). We observed
79 that capsule enlargement is coordinated with cell cycle progression. Therefore, and
80 given that *cln1* was unable to melanize, we decided to study the relationship between
81 this particular cyclin of *C. neoformans* and the ability of the yeast to produce melanin.
82 Our results demonstrate that the production of melanin seems to be regulated through a
83 complex pathway in which cell wall stability is essential. In addition, drugs that cause
84 cell cycle arrest in G1/S phase results in defects in melanization.

85

86 **Materials and methods**

87 **Strains and culture conditions**

88 *Cryptococcus neoformans* var. *grubii* H99 strain (Perfect et al., 1980) and the mutant
89 CNAG_06092 (*cln1*) obtained from a collection of mutants deposited at the ATCC by
90 Dr. Madhani (Liu et al., 2008) were used in this study. In addition, the reconstituted
strain, *cln1::CLN1* was generated by biolistic transformation as described in (Garcia-Rodas et al., 2014)

The strains were routinely grown in liquid Sabouraud medium (Oxoid LTD, UK) at 30°C or 37°C with moderate shaking (150 r.p.m). To induce melanization, strains were grown protected from light in chemically defined minimal medium (15 mM dextrose, 10 mM MgSO₄, 29.4 mM KHPO₄, 13 mM glycine and 3 µM thiamine, pH 5.5) with 1 mM L-DOPA (Sigma-Aldrich, St. Louis, USA) and incubated at 30°C with shaking. In some cases, the same medium without L-DOPA was used as a control.

For solid media, 1.5% agar was added to the medium. Yeast suspensions were prepared at 2x10⁷/mL in PBS. Serial 1:10 dilutions were performed and 5 µL from each dilution were spotted. Plates were incubated at 30 and 37°C and pictures were taken daily. In some cases, 1M Sorbitol or Congo Red (0.05; 5 and 10 mg/mL) were added to Sabouraud agar plates.

## Phylogenetic tree

We performed evolutionary studies of the Cln1 protein sequence using PhylomeDB (http://phylomedb.org) (Huerta-Cepas et al., 2008; Huerta-Cepas et al., 2011) by Dr. Gabaldón's lab (Comparative Genomics Group at Centre for Genomic Regulation, Barcelona, Spain). PhylomeDB is an algorithm that allows the visualization of the evolution of a specific gene by comparison of multiple genomes. After introduction of the protein sequence, the software performs multiple alignments and creates a tree where the evolution of a specific protein (duplication and speciation events) can be visualized and paralogs and orthologs identified.

## Wheat germ agglutinin staining
The presence of chitin-like structures, WGA staining was ascertained as described in (Rodrigues et al., 2008a). Briefly, *C. neoformans* cells with enlarged capsule (incubated in 10% Sabouraud in 50 mM MOPS buffer pH 7.3 overnight at 30°C) (Zaragoza and Casadevall, 2004) of *C. neoformans* strains were washed in PBS and suspended in 4% p-formaldehyde for 30 min at room temperature. The fixed cells were washed in PBS and suspended in 100 µL of a 5 µg/mL of WGA conjugated to Alexa-594 (Molecular Probes, Invitrogen) for 1 h at 37°C. After incubation, cells were washed with PBS and suspended in 100 µL of PBS. Cell suspensions were mounted over glass slides and photographed with a SP5 confocal microscope (Leica Microsystems).

## 123 Melanin production

Melanin production was assessed on both liquid and solid chemically defined minimal medium (see above) supplemented with L-DOPA. After 24-48 h, melanization was assessed by acquisition of a dark color of the cultures, compared to parallel cultures without L-DOPA. For melanin production on solid medium, yeast suspensions were prepared at 2x10⁷/mL in PBS. Serial 1:10 dilutions were performed and 5 µL from each one were spotted on L-DOPA plates and on Sabouraud dextrose agar. Plates and cultures were incubated at 30 and 37°C protected from light. Pictures of either the plates or liquid cultures were taken daily.

## 132 Laccase activity assay

*Cryptococcus neoformans* strains were incubated in 15 mL of chemically defined minimal medium with L-DOPA overnight at 30°C with shaking (150 r.p.m) and protected from light. Laccase activity was assessed as described in (Alvarado-Ramirez et al., 2008) with some modifications. Pelleted cells were suspended in 2 mL of PBS and divided in two equal fractions; one was used as a negative control by inactivating the laccase activity with 5% β-mercaptoethanol for 2 h at 37°C. Then, both aliquots
139 were disrupted using a Fast Prep homogenizer (MP Biomedicals) with 500 µL of 425-
140 600 µm Ø glass beads (Sigma-Aldrich, St. Louis, USA). A minimum of 6 cycles of 45 s
141 were performed (with intervals of 4 min in ice). The mixture was centrifuged at 13,800
g for 10 min at 4°C. Supernatants were conserved at 4°C until enzymatic determination
142 was performed. To quantify the laccase activity, 100 µL of every sample were placed in
143 a 96 well-plate (Costar, NY, USA), and 7 µl of a 20 mM L-DOPA solution were added
144 to each well. The microplate was incubated at 25°C with moderate shaking in an iEMS
145 Spectrophotometer (Thermofisher) for 18 h. Optical density at 450 nm was measured
146 every 15 min. Protein concentration of the extracts was determined with the Bradford
147 method using the Quick Start Bradford Protein Assay (BioRad, CA, USA). Specific
148 activity was expressed as mUAbs/min/µg protein.
149

150

151 **Quantification of the expression of the LAC1 gene by Real-time PCR.**

152 Yeast cells were grown for three days in minimal medium at 30°C with shaking (150
153 rpm) as described above. RNA extraction was performed using Trizol reagent protocol
154 (Ambion RNA by life technologies) with some modifications. The cells were disrupted
155 with glass beads using a FastPrep-24 (MPTM) for 5 minutes, alternating 20 seconds
156 shaking with 1 minute on ice. The RNAs were quantified and qualified using the
157 Nanodrop 8000 Spectrophotometer (Thermo scientific). cDNAs were generated using
158 the iScripTM cDna synthesis Kit (BioRad) following the manufacturer's
159 recommendations. The RT-PCR was performed using SsoAdvancedTM Universal
160 SYBR® Green Supermix (BioRad) using the LAC1 specific primers (LAC1F
161 AGAAGGGAAGGAAGGTGATG and LAC1R1 TATACCTCACAACCGCCAAT
162 described in (Alanio et al., 2011) in a total volume of 20 µl, in a Light Cycler® 480
163 (Wang et al.). As control, 18s specific primers were used (18sF
164 CCGTTGCTAGAGGTGAAATTCTTAG and 18sR
165 ATCTAATCGTTTTTGATCCCCTAAC). The RT-PCR conditions were 95°C for 10
166 minutes and 40 cycles of amplification (95°C for 15 s, 58°C for 1 minute). Differences
167 in gene expression were calculated using the 2<sup>ΔΔCt</sup> method.

168 Susceptibility to oxidative stress

169 *Cryptococcus neoformans* strains were incubated in L-DOPA liquid medium for 48 h at
170 30°C with shaking (150 r.p.m). Suspensions were prepared at 2x10³ cells/mL in PBS.
171 Hydrogen peroxide (Fluka, St. Louis, USA) was added at different final concentrations
172 (0.5, 1 and 2 mM) and cells were incubated at 30°C for 1h. Samples without hydrogen
173 peroxide were incubated in parallel as controls. Each sample was carried out in
174 triplicate. Then, 50 and 100 µL were plated on Sabouraud agar plates and incubated at
175 30°C for 2 days. The number of colony forming units (CFU) was enumerated, and
176 viability was calculated as the percentage of colonies obtained in the treated samples
177 compared with the untreated controls.

178 Melanin production in the presence of cell cycle inhibitors

179 *Cryptococcus neoformans* strain H99 cells were grown in 5 mL of Sabouraud at 30°C
180 with agitation. Cells were washed and transferred to L-DOPA liquid medium containing
181 different rapamycin concentrations (0.5 and 1 µg/mL, Sigma Aldrich, St. Louis, USA),
182 which induces G1 arrest. The same amount of DMSO (Sigma Aldrich, St. Louis, USA)
183 was added to the control cultures (without the drug) since rapamycin is diluted in this
184 solvent. Cells were grown overnight at 30°C and pictures were taken daily to asses
185 melanization.

186 To assess cell viability after treatment with the different concentrations of rapamycin
187 cells were stained with propidium iodide at a final concentration of 5 µg/ml and
quantified by flow cytometry using a FacsCalibur flow cytometer (BD, Biosciences, 188
Worburn, MA). Data were processed using CellQuest (BD, Biosciences) and FlowJo 189
 national (Tree Star Inc, Ashland, OR) softwares. 190

Growth curves in L-DOPA liquid medium in the presence of cell cycle inhibitors
191

Suspensions of *Cryptococcus neoformans* strain H99 at 5x10⁵/mL were prepared in
chemically defined minimal medium with 1 mM L-DOPA containing different
concentrations of rapamycin (0, 0.5 or 1 µg/mL). Control suspensions of *C. neoformans*
cells in chemically defined medium with DMSO were carried out in parallel in each
case. Cells were grown for 48 h at 30°C and optical density at 540 nm was assessed
every hour using an iEMS Spectophotomer (Thermofisher). Graphs were plotted using
Graph Pad Prism 5. 198

199

200 Statistical analysis

Scatter plot graphs of cell sizes were done using Graph Pad Prism 5 (La Jolla, CA, 201
USA), and statistical differences were assessed with T-test. A *p* value <0.05 was 202
considered significant. 203

204

205 Results

206 Phylogenetic analysis of Cln1

Previous work of our laboratory has focused on the role of Cln1 on capsule production
(Garcia-Rodas et al., 2014). During the characterization of the role of this protein on *C.
neoformans* capsule formation, we found that the corresponding mutant strain presented
some abnormal phenotypes that were not expected to be related with cell cycle 207
208
209
210
regulation, such as a defect in melanization. In consequence, we decided to further
characterize phenotypically this mutant. First, we performed a blast comparison using
PhylomeDB, an algorithm that allows the generation of phylogenetic evolutionary trees
and the identification of gene paralogs and orthologs. The results showed that although
Cln1 had evolutionary similarities with cyclins from other organisms, that in C.
neoformans, this cyclin had evolved specifically with proteins present only in other
basidiomycetes after two duplication events from a common ancestor (Figure 1).
Therefore, it is possible that this protein has new roles in C. neoformans development
and/or pathogenesis independently of cell cycle functions.

220 **Growth and morphological characteristics of cln1 mutant**

221 We investigated the growth rate of *cln1* at different temperatures. The mutant *cln1*
222 showed growth defects at 37°C in rich medium Sabouraud (Figure 2). However, this
223 defect was partially restored when 1M Sorbitol was added (Figure 2). Interestingly,
224 sorbitol did not restore other morphological defects described for this mutant, such as an
225 engrossment of the bud neck or enlarged cell size (data not shown). This result
226 suggested that *cln1* had cell wall defects. Therefore, we investigated if *cln1* was more
227 susceptible to agents that affect the stability of the cell wall, such as Congo Red. As
228 shown in figure 3, Congo Red inhibited *cln1* growth in a dose dependent manner, which
229 was linked to possible cell wall alterations. We tested other cell wall disturbing
230 compounds, such as tunicamycin, or osmotic stress (NaCl), but we did not find any
231 difference in the growth between any of the strains.

232 Chitin-like molecules in fungi are polymerized by chitin synthases, which use
233 cytoplasmic pools of UDP-GlcNAc (N-acetylglucosamine) to form β-1,4-linked
234 oligosaccharides and large polymers. In C. neoformans, the final cellular site of chitin
accumulation is the cell wall, although some protuberances of this structure have been
also found in the capsule (Rodrigues et al., 2008a). The chitin-like structures in the
capsule can be visualized by the binding of fluorescent wheat germ agglutinin, which
binds to sialic acids and β-1,4-N-acetylglucosamine (GlcNAc) oligomers (Rodrigues et
al., 2008a). We used WGA to ascertain whether these structures were also present in the
*cln1* mutant. Cells from the wild type and *cln1::CLN1* strains bound WGA at the neck
between the mother cell and the bud. In contrast, in cells from the *cln1* strain, these
structures were delocalized and were found all over the cell giving also a more intense
fluorescent signal (Figure 4).

## 244 Melanin production and laccase activity

During the phenotypic characterization of *cln1*, we observed that Cln1 was required for
the accumulation of melanin (Figure 5A). Wild type and *cln1::CLN1* strains produced
melanin after 3-4 days of incubation at 30°C on L-DOPA agar plates (Figure 5A) while
*cln1* mutant was unable to accumulate melanin. In liquid medium, we found that
melanization of the wt and *cln1::CLN1* strains was visible after 24 h of incubation with
L-DOPA, while the *cln1* mutant did not accumulate melanin.

To get insights about this phenotype, we measured the laccase activity in the wild type
and mutant strains (Zhu et al., 2001). In agreement with the visual observation showing
absent melanization, the *cln1* mutant had no laccase activity in the conditions tested.
This defect was recovered in the reconstituted strain (Figure 5B).

We then investigated if the defect in laccase activity was due to a defect at the
transcriptional level. We measured the relative levels of the mRNA of the *LAC1* gene
by real-time PCR. For this purpose, total RNA was isolated from cells grown in
minimal medium and cDNA was obtained using standard protocols. We then used this
cDNA as template to quantify the levels of the *LAC1* mRNA. The expression of the
260 LAC1 gene in the *cln1* mutant was significantly lower than in the WT strain, and this
defect was partially complemented in the reconstituted strain (figure 5C), indicating that
261
absence of melanization was mainly due to a defect in the transcription of the *LAC1*
262
263 gene.

264

265 Melanin protects *C. neoformans* from oxidative damage

266 Laccase and melanin confer resistance to oxidative damage (Wang et al., 1995), and
consequently we tested the susceptibility of Cln1 sufficient and deficient strains to
267
$H_2O_2$. Cells from WT and *cln1::CLN1* grown for 48 h in L-DOPA liquid medium
268
showed resistance to oxidative stress at all concentrations tested (0.5, 1 and 2 mM,
269
Figure 6). In contrast, *cln1* cells grown in the same conditions showed a five-fold
270
reduction in survival when exposed to $H_2O_2$, even when using low $H_2O_2$ concentrations
271
such as 0.5 mM (Figure 6). In contrast, *cln1* and WT cells grown in Sabouraud liquid
272
medium showed no statistical significant differences in survival when incubated with
273
different concentrations of $H_2O_2$ (data not shown).
274

275 Melanin production in the presence of cell cycle inhibitors

276 The lack of melanization in *cln1* suggested a link between cell cycle elements and the
capability to produce and/or accumulate melanin. Therefore we examined if melanin
277
production was affected by drugs that cause cell cycle arrest. First, we tested
278
melanization in L-DOPA liquid cultures containing different concentrations of
279
rapamycin (0.5 and 1 µg/mL), which is an inhibitor of the TOR signaling pathway and
280
causes G1 arrest. As shown in figure 7, rapamycin inhibited melanin production by *C.
281
neoformans* in L-DOPA liquid cultures (Figure 7A). To assess if lack of melanization
282
283 was due to an inhibitory effect of rapamycin on growth rate, we studied growth in L-
284 DOPA liquid medium with different concentrations of rapamycin and observed that this
285 drug had no inhibitory effect on the growth rate of C. neoformans in L-DOPA liquid
286 medium (Figure 7B).

287

288 **Discussion**

289 During the phenotypic characterization of the *cln1* mutant, we found that it was unable
290 to synthesize melanin in the presence of substrates such as L-DOPA. The production of
291 melanin is known to confer protection against different stress conditions (Jacobson and
292 Tinnell, 1993; Emery et al., 1994; Wang and Casadevall, 1994a;b; Rosas and Casadevall,
293 1997). Some factors that regulate melanization have already been described (Alspaugh
294 et al., 1997; 1998; D'Souza et al., 2001; Nosanchuk et al., 2001; Vidotto et al.,
295 2002; Tangen et al., 2007; Mauch et al., 2013). In *Cryptococcus neoformans* this process
296 only takes place in the presence of specific substrates, mainly diphenolic compounds
297 (Chaskes and Tyndall, 1975; 1978). Melanization depends on the expression of a gene
298 that encodes a diphenol oxidase, called laccase, and the expression of this gene is
299 repressed by glucose (Nurudeen and Ahearn, 1979). Our initial finding suggested that
300 melanization is in fact under the regulation of a more complex circuit, and for that
301 reason, we decided to characterize this phenotype in detail.

302 Consistent with its role in cell cycle regulation, the cyclin mutant displayed growth
303 defects, especially at 37°C. This might be explained by the fact that some genes that are
304 expressed at the G1/S phase boundary are involved in cell wall biosynthesis (Igual et
305 al., 1996). This idea is supported by the restoration of the growth defect phenotype after
306 the addition of osmotic stabilizers (sorbitol) to the media and the higher susceptibility to
307 agents that alter the stability of the cell wall such as Congo Red. We tested if the *cln1*
308 mutant had any defect in the activation of MAPK that are involved in the response to
309 cell wall alterations (such as Mkc1 and Cek1) (Navarro-Garcia et al., 1995; Navarro-
310 Garcia et al., 2005; Eisman et al., 2006; Roman et al., 2009), but our preliminary data
311 showed no difference in the activation of these kinases between any of the strains tested
312 in this work (data not shown).

313 Laccase in *C. neoformans* is synthesized in cytoplasm (Jacobson, 2000; Garcia-Rivera et
314 al., 2005; Eisenman et al., 2007) and then it is believed to be transported in vesicles to
315 the cell wall, where it is finally tightly linked through disulfide or thioester bond. (Zhu
316 et al., 2001; Waterman et al., 2007; Rodrigues et al., 2008b) The mutant *cln1* presents cell
317 wall defects and miss localization of chitin like structures as evidenced by the
318 delocalized binding of WGA. Therefore, we believe that laccase is not correctly linked
319 to cell wall and thus, the *cln1* mutant strain is unable to produce melanin. This
320 phenotype was also confirmed independently by testing the susceptibility of *cln1* to
321 stress factors, such as H2O2, and the effect of cell cycle inhibitors on melanization.

322 Curiously, we observed that *cln1* could grow normally at 37°C in minimal medium.
323 Growth curves performed with *C. neoformans* WT strain in the presence of cell cycle
324 inhibitors in minimal medium revealed that rapamycin had no effect on growth rate.
325 These results suggest that neither rapamycin, nor Cln1 regulate growth rate in minimal
326 medium, although they are necessary to melanize. Cln1 has been involved in the
327 regulation of cell cycle in *C. neoformans* (Virtudazo et al., 2010; Virtudazo et al.,
328 2011; Garcia-Rodas et al., 2014). However, our findings suggest that in certain
329 conditions, cell cycle progression does not depend on Cln1 nor Tor proteins, and these
330 proteins seem to have other functions in other metabolic processes, such as melanin
331 production, independently of cell cycle regulation.

332 Finally, our results show a plausible link between cell cycle and melanin production.
333 The phylogenetic analysis showed that Cln1 had evolved differently compared to
cyclins of other microorganisms, and that this evolutionary pattern is only present in basidiomycetes. This result suggests that Cln1 of *C. neoformans* has acquired specific functions that are not present in other microorganisms, and thus, it could regulate processes, such as melanin production, characteristic of *C. neoformans*. Cln1 modulates G1/S transition in *C. neoformans* and its absence leads to defects on virulence determinants, such as melanin production. This study offers new directions in cryptococcal virulence research and the possibility of new drug targets involved in cell cycle progression.

**Acknowledgements.** We thank Rajendra Uphadya (Washington University School of Medicine, St. Louis) for providing the sequence of oligonucleotides for 18s gene used in this article. R.G.-R. was supported by a FPI fellowship (reference BES-2009-015913) from the Spanish Ministry of Economics and Competitivity. N.T-C. is supported by a FPI fellowship (reference BES-2012-051837). O.Z. is funded by grant SAF2011-25140 from the Spanish Ministry for Economics and Competitivity.

348

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517

518

519

520 **Figure legends**

521 **Figure 1. Phylogenetic tree of Cln1.** A phylogenetic tree analysis of *C. neoformans*

522 Cln1 protein sequence was performed using PhylomeDB as described in Material and

523 Methods. The sequence of *C. albicans* Ccn1 was used as root. The location of the *C.*

524 *neoformans* Cln1 sequence is highlighted with a black arrow. Red dots indicate

525 duplication events, and in consequence, appearance of paralogs, and blue dots denote

526 speciation events and appearance of orthologs.

527 **Figure 2. Growth of cln1 mutant on solid medium.** Panels show fungal growth on

528 Sabouraud agar plates, Sabouraud agar plates supplemented with 1M Sorbitol, at 30°C

529 (upper panels) and at 37°C (lower panels). Upper numbers indicate the number of cells

530 placed in each spot. Pictures were taken after 48h.

531 **Figure 3. Cell wall stability assay.** Growth of wt, *c1n1* and the reconstituted strains on

532 Sabouraud agar plates with different Congo Red (CR) concentrations. Plates were

533 incubated at 30°C and pictures were taken after 48h.

534 **Figure 4. Binding of wheat germ agglutinin to C. neoformans strains.** Panels show

535 representative cells of each strain grown in capsule inducing medium at 30°C for 24 h

536 stained with WGA as described in M&M.
**Figure 5. Melanin production and laccase activity.** (A) growth of *C. neoformans* strains on L-DOPA agar plates at 30°C. (B) Laccase activity of *C. neoformans* strains. A *p* value < 0.05 is indicated with an asterisk. (C) **LAC1** gene expression. Cells from WT, *cln1* and *CLN::CLN1* strains were grown in minimal media for three days at 30°C and RNA samples were isolated. cDNAs were obtained and relative expression of the *LAC1* gene was measured by real time PCR.

543

**Figure 6. Susceptibility to oxidative stress.** Bars show different susceptibility of *C. neoformans* strains grown in L-DOPA liquid medium and incubated in the presence of different H₂O₂ concentrations for 1 hour.

**Figure 7. Growth curves under cell cycle inhibitors and their effect on melanization.** (A) Panels show cultures of *C. neoformans* in L-DOPA liquid medium containing different rapamycin concentrations at 0 and 48 hours. Cultures with DMSO were carried out in parallel since rapamycin was dissolved in DMSO. (B) Growth of *C. neoformans* in L-DOPA medium containing different rapamycin concentrations. Optical density (O.D.) was measured at 540 nm every hour for 48 hours. Black line, growth in control medium (minimal medium + DMSO); red line, rapamycin 0.5 µg/mL; green line, rapamycin 1 µg/mL.

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