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Developmental cell fate and virulence are linked to trehalose homeostasis in 15 Cryptococcus neoformans 16
Michael R. Botts1,†, Mingwei Huang1, Regen K. Borchardt1¥, Christina M. Hull1,2,* 17 18 19
1Department of Biomolecular Chemistry, 2Department of Medical Microbiology and Immunology, 20 School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53706 21
22 23 24 25 26 27 *Address correspondence to: 28 Christina M. Hull, PhD 29 1135 Biochemistry Building 30 420 Henry Mall 31 Madison, WI 53706 32 Telephone: (608) 265-5441 33 Fax: (608) 262-5253 34 E-mail: cmhull@wisc.edu 35 36 †Current Address: 37 Division of Biological Sciences 38 University of California- San Diego 39 9500 Gilman Dr. #0349 40 4205 Bonner Hall 41 La Jolla, CA 92093-0349 42 43 ¥Current Address 44 Cellular Dynamics International, Inc. 45 525 Science Drive Suite 100 46 Madison, WI 53711 47 48 Keywords: Cryptococcus neoformans, spores, sexual development, trehalose, fungal 49 pathogenesis 50
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EC Accepts, published online ahead of print on 7 July 2014Eukaryotic Cell doi:10.1128/EC.00152-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Abstract 52
Among pathogenic environmental fungi, spores are thought to be infectious particles that 53
germinate in the host to cause disease. The meningoencephalitis-causing yeast Cryptococcus 54
neoformans is found ubiquitously in the environment and sporulates in response to nutrient 55
limitation. While the yeast form has been studied extensively, relatively little is known about 56
spore biogenesis, and spore germination has never been evaluated at the molecular level. 57
Using genome transcript analysis of spores and molecular genetic approaches we discovered 58
that trehalose homeostasis plays a key role in regulating sporulation of C. neoformans, is 59
required for full spore viability, and influences virulence. Specifically, we found that genes 60
involved in trehalose metabolism, including a previously uncharacterized secreted trehalase 61
(NTH2), are highly overrepresented in dormant spores. Deletion of the two predicted trehalases 62
in the C. neoformans genome, NTH1 and NTH2, resulted in severe defects in spore production, 63
a decrease in spore germination, and an increase in the production of alternate developmental 64
structures. This shift in cell types suggests that trehalose levels modulate cell fate decisions 65
during sexual development. We also discovered that deletion of the NTH2 trehalase results in 66
hypervirulence in a murine model of infection. Taken together, these data show that the 67
metabolic adaptations that allow this fungus to proliferate ubiquitously in the environment play 68
unexpected roles in virulence in the mammalian host and highlight the complex interplay among 69
the processes of metabolism, development, and pathogenesis. 70
71
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Introduction 72
A fundamental requirement for organisms to survive is the ability to adapt to changes in the 73
availability of metabolic fuels. Metabolic acclimation enhances survival in a given environment 74
or provides the capacity to exploit a new one. In response to nutrient limitation, organisms 75
generally decrease their growth rates; however, when nutrients become severely limiting, many 76
microbes undergo a transition from active growth to dormancy (1). In some bacteria and fungi, 77
this transition results in the development of a new cell type, the spore, and is coupled with 78
structural and metabolic adaptations that allow spores to remain dormant and withstand severe 79
environmental stresses (2–4). Once spores encounter more favorable growth conditions, they 80
undergo another transition (germination) to resume vegetative growth (5). 81
For environmental human fungal pathogens, spores are likely infectious particles (6–9). 82
The properties of spores that allow them to withstand harsh conditions and disperse throughout 83
the environment (e.g. small size, tolerance for high temperatures, resistance to oxidative stress, 84
etc.) also allow them to encounter hosts and initiate disease-causing infections. This is true of 85
Cryptococcus neoformans, a human fungal pathogen for which both yeast and spore forms are 86
plausible infectious particles (10). C. neoformans spores are produced on aerial fruiting bodies 87
(basidia) allowing for dispersal, which along with their resistance to external stresses and ability 88
to cause disease in a murine model of infection, has led to the hypothesis that C. neoformans 89
spores are natural infectious particles (11, 12). C. neoformans infects people from 90
environmental sources through a respiratory route in which the organism is inhaled and 91
proliferates within the lung. In immunocompromised individuals, such as people with AIDS, this 92
infection can disseminate to the central nervous system and cause meningoencephalitis (13). C. 93
neoformans spores, also known as basidiospores, are produced during sexual development 94
that occurs in response to nutritional signals and involves the formation of several new cell 95
types that reside in multicellular structures. While the morphological changes that occur during 96
sexual development have been described, relatively little is known about the metabolic signals 97
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or molecular pathways that influence cell fate decisions and ultimately lead to the production of 98
spores and their ability to germinate in new environments. 99
To begin to understand spore biogenesis and germination, we carried out an analysis of 100
spore transcripts in both dormant spores and a germinating population of spores. Transcripts of 101
genes important for trehalose metabolism were highly overrepresented in dormant spores, 102
suggesting a role for the sugar trehalose in C. neoformans spore biology. Trehalose is a 103
disaccharide of two -linked glucose molecules (-1,1-glucose) that has been broadly linked to 104
stress resistance and development in many organisms (14–18). In several fungi trehalose 105
biosynthesis has been intimately linked to virulence, morphogenesis, and production of viable 106
spores. In the human pathogen, Candida albicans, trehalose biosynthesis is required for 107
resistance to oxidative stress, and trehalose metabolism has been linked to both stress 108
resistance and the yeast to hyphal transition, which are both important for virulence. Trehalose 109
synthesis mutants of C. albicans also show decreased virulence (19–22). Similarly, trehalose 110
has well established roles in both virulence and spore production in the filamentous fungal 111
pathogen, Aspergillus fumigatus, in which trehalose synthesis is important for general stress 112
response and the production of robust, stress-resistant spores (23). In contrast to other fungal 113
pathogens, deletion of the trehalose biosynthesis genes in A. fumigatus, TPSA and TPSB, 114
resulted in increased virulence in a murine model of invasive aspergillosis, likely caused by 115
alterations in the cell wall making mutant hyphae resistant to phagocytosis by macrophages 116
(23). Taken together, these findings reveal a complex and diverse role for trehalose metabolism 117
in regulating the physiology of fungal pathogens. 118
In C. neoformans disruption of either of the genes involved in trehalose synthesis (TPS1 119
or TPS2) results in severe defects in both sexual development and virulence, but the 120
mechanisms by which trehalose controls these functions are not known (24–26). Previously, a 121
single trehalose-degrading enzyme NTH1 (Neutral TreHalase 1) was studied, and surprisingly, 122
deletion of this gene caused no observable phenotypes (25). Through our gene expression 123
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analysis of spores, we discovered a second predicted trehalose-degrading enzyme that we 124
have designated NTH2 (Neutral TreHalase 2) that was overrepresented within dormant spores. 125
The discovery of this trehalase allowed us to assemble a more complete picture of 126
trehalose metabolism and the relationships between trehalose synthesis and breakdown during 127
spore formation, dormancy, and germination. We found that trehalase mutants harbor defects in 128
sexual development including, aberrant fruiting body (basidium) formation, a decrease in spore 129
formation, and decreased spore viability. We also observed that aberrant basidium formation 130
was accompanied by an increase in the production of apparent chlamydospores, a previously 131
described alternate developmental cell type (26), thus implicating trehalose as a signaling 132
molecule in C. neoformans cell fate determination. Finally, we determined that the absence of 133
NTH2 led to a significant increase in virulence in a murine model of infection, suggesting that 134
the trehalose catabolic pathway is active in the mammalian host. Overall, our results indicate 135
that trehalose metabolism is a core component of both sexual development and pathogenesis 136
and underscore the importance of trehalose homeostasis in these diverse processes in C. 137
neoformans. 138
139
Materials and Methods 140
Strains and media. All strains used were of the serotype D background (Cryptococcus 141
neoformans var. neoformans strains JEC20 (a) and JEC21 ()) (27). All were handled using 142
standard techniques and media as described previously (28). Sexual development assays were 143
conducted on V8 agar medium pH 7.0 at 22°C in the dark for 2-7 days. 144
145
Microscopy. All micrographs were created using an Axio Scope.A1 with long working-distance 146
objectives and an Axiovision MRM camera running Axiovision software. 147
148
RNA isolation and amplification. Spores were isolated by scraping sexually developing 149
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populations of cells from V8 agar and suspending in 65% Percoll made isotonic with Phosphate 150
Buffered Saline (PBS). Suspensions were subjected to centrifugation (10,000 x g, 30 minutes, 151
4oC), and spores were collected from the bottom of the gradient and counted. A 4 x 108 spore 152
aliquot was frozen in liquid N2 to serve as the zero time point sample. The remaining spores (1.6 153
x109) were resuspended in 6 mL of liquid YPD and incubated at 30°C with shaking at 200 RPM. 154
Samples were collected every 2 hours for 10 hours and frozen in liquid nitrogen. RNA was 155
extracted from frozen spores using hot acid-phenol as described previously and was further 156
purified using the RNEasy Mini Kit (Qiagen, Valencia CA) (29). RNA amplification was carried 157
out using the MessageAmp II amplification kit (Ambion) according to manufacturer 158
specifications. 500 ng of total RNA from each germinating time point was used as the starting 159
material. After production of antisense RNAs containing aminoallyl-UTPs (aRNAs), each sample 160
was assessed for quantity and average length of aRNA using an RNA Pico Chip and the Agilent 161
Bionalyzer 2100 (Agilent Technologies, Santa Clara CA). Before hybridization, equal amounts of 162
aminoallyl-UTP incorporated aRNA were conjugated to Oyster dyes (Boca Scientific) according 163
to manufacturer instructions. 164
165
Microarray hybridization and analysis. Fluorescently labeled aRNAs were hybridized to C. 166
neoformans whole genome spotted microarrays of 70-mer oligonucleotides containing 167
7765 open reading frame (ORF) probes. The data presented for each experiment represent 168
eight-fold coverage of ORFs: quadruplicate experiments (including dye-swap), with each slide 169
containing ORF probes spotted in duplicate. Slides were incubated as reported previously (30). 170
Extracted data were analyzed using the GeneSpring 10.0 software package (Agilent 171
Technologies, Santa Clara CA). Data were normalized using the LOWESS 172
(locally weighted scatter plot smoothing) algorithm and assessed for statistical significance 173
utilizing ANOVA (31). Two thousand five hundred and twelve genes meeting the 174
criterion p<0.05 were considered statistically significant and used in further analyses (32). 175
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Significant genes meeting a two fold-change threshold between any two time points (1,499) 176
were clustered using Cluster 3.0. Clusters were assessed for Kyoto Encyclopedia for Genes 177
and Genomes (KEGG) pathway, and Gene Ontology (GO) term enrichment using the Database 178
for Annotation, Visualization, and Integrated Discovery (DAVID) (33–35). The full dataset has 179
been deposited in the NCBI Gene Expression Omnibus database (GEO) and can be accessed 180
through GEO accession number GSE53912 (36). 181
182
Generation of deletion strains. Gene deletion constructs were created using fusion PCR (37). 183
The NTH1 5'- region was amplified with primers CHO1565 and CHO1566, and the 3'-region was 184
amplified with CHO1567 and CHO1568. The NATR cassette was amplified with CHO1569 and 185
CHO1570. PCR fusion using CHO1565 and CHO1568 was used to create the final nth1::NATR 186
deletion cassette. The NTH2 5'- region was amplified with primers CHO2636 and CHO2637, and 187
the 3'-region was amplified with CHO2638 and CHO2639. The NEOR cassette was amplified 188
with CHO2640 and CHO2641. PCR fusion using CHO2636 and CHO2639 was used to create 189
the final nth2::NEOR deletion cassette. The nth1::NATR and nth2::NEOR deletion cassettes were 190
transformed into JEC20 and JEC21 by biolistic transformation, grown on medium containing 1 M 191
sorbitol, and selected on medium containing 200 µg/mL G418 or 200 µg/mL nourseothricin (38). 192
Resulting colonies were screened using PCR for correct insertion of the knockout construct and 193
assessed via Southern blotting for single integration of the construct to identify multiple 194
independent knockouts (29). Each mutant was then crossed with wild type yeast to obtain 195
knockout progeny in both mating types. The resulting F1 progeny were then rescreened by PCR 196
for the absence of the deleted gene to confirm the strain genotype. Double mutants, nth1 197
nth2, were constructed by backcrossing a nth2 (CHY2082) with nth1 (CHY1710), isolating 198
and germinating spore progeny from this cross, and then replica plating these progeny onto 199
solid medium containing both nourseothricin and G418. Double mutants were then rescreened 200
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by PCR to confirm the absence of NTH1 and NTH2 open reading frames (CHY2184). 201
202
NTH1 and NTH2 transcript analysis. RNA was prepared from C. neoformans yeast or crosses 203
as described previously (29). Northern blots were conducted according to standard protocols 204
using 10 µg total RNA per sample. Probes were generated by PCR amplification utilizing the 205
ExTaq PCR system (TaKaRa Bio Inc.) and oligos CHO2979 and CHO2980 (NTH1), CHO2938 206
and CHO2939 (NTH2) and CHO1696 and CHO1697 (GPD1). Probes were radiolabeled using 207
Ready-To-GoTM DNA Labeling Beads (GE Healthcare LifeSciences, Piscataway, NJ) according 208
to manufacturer’s instructions and hybridized to blots at 65ºC as described previously (39). Blots 209
were exposed to a phosphor screen and imaged with a Storm 860 PhosphorimagerTM 210
(Molecular Dynamics, GE Amersham). 211
212
Quantification of trehalose 213
Trehalose was quantified in C. neoformans crosses using a colorimetric enzyme linked 214
assay previously used in S. cerevisiae (40). Crosses were spotted on V8 for 5 days, harvested 215
into 100 mM sodium citrate pH 5.7, and boiled for 10 minutes. The resulting suspensions were 216
lysed using sonication on a 30% duty cycle at level 2 for five minutes, and lysis was confirmed 217
microscopically. Insoluble cell debris was pelleted and discarded. Lysates (100 µL) were treated 218
with 1U of porcine kidney trehalase (Sigma Aldrich) for 4 hours at 37°C. Glucose produced was 219
quantified using a glucose assay kit (Sigma Aldrich) according to manufacturer's instructions. 220
Absorbance at 540nm values for lysates was compared to a standard curve of glucose and 221
normalized to the starting mass of the cell pellet. Calculated trehalose levels were subjected to 222
one-way ANOVA comparing each sample to an a x cross. 223
224
Phenotype testing. A battery of common phenotypes was assessed, including melanin 225
production, capsule production, growth at 37°C, and cell wall integrity. In each case, wild type, 226
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nth1, nth2, and nth1 nth2 strains were evaluated. Melanin production: grew cells on L-227
DOPA agar at 37°C for 3 days and visually assessed the production of pigment . Capsule 228
production: cultured strains in Dulbecco’s modified eagle medium (DMEM) with 5% fetal calf 229
serum at 37°C in 5% CO2 (41) for two days then stained with India ink and assessed 230
microscopically (42). Growth at 37°C: spotted serial dilutions of cells onto YPD agar, incubated 231
at 37°C for two days, and assessed visually (25). Cell wall integrity: spotted serial dilutions of 232
cells onto medium containing calcofluor white MR2, sodium dodecyl sulfate, or caffeine and 233
grew for two days at 37°C (43). Resistance to heat shock: grew cells on V8 agar pH 7.0 at 234
25°C for 2 days, suspended in PBS, and incubated at 42°C and 50°C for 5, 10, 15, and 30 235
minute intervals, diluted, and plated to YPD for 3 days at 30°C (3). Resistance to oxidative 236
stress: spread 1 x 106 yeast onto 100 mm diameter V8 agar plate with a 6 mm disc of Whatman 237
paper containing 10 µL 10% H2O2 in the center and incubated at 30°C for 3 days. (44). 238
Germination frequencies were determined by carrying out crosses on V8 for 5 days and 239
isolating spores. Spores were counted, plated onto rich medium, and allowed to germinate and 240
grow at 30°C for 3 days. Germination frequencies were determined by dividing the number of 241
colonies formed by the number of spores plated. Three replicates were performed for each 242
strain, and the mean for each strain was compared to wild type using the Student’s t-test. 243
244
Virulence analysis. Strain virulence was assayed using a murine intravenous model of 245
infection (45). Wild type nth1, nth2, and nth1 nth2 yeast were grown to mid-log 246
phase and washed three times in phosphate buffered saline (PBS). Five female Balb/c mice 247
were inoculated via tail vein with 1 x 106 yeast of each strain. Only the yeast cell type was used 248
to infect the mice because we could not recover sufficient numbers of mutant spores to carry out 249
spore-mediated infections. The mice were observed daily for rapid weight loss or signs of pain 250
at which point moribund mice were euthanized as described previously (12). The resulting 251
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survival times were plotted on a Kaplan-Meyer plot, and median survival times of mice were 252
analyzed using a Wilcoxon rank-sum test. This experiment was carried out twice with 253
independently derived mutant strains, and the results were identical between experiments. The 254
animal studies were reviewed and protocol approved by the University of Wisconsin- Madison 255
Institutional Animal Care and Use Committee (Protocol A01506). The University of Wisconsin- 256
Madison is AAALAC accredited, and the facilities meet and adhere to the standards in the 257
“Guide and Care of Laboratory Animals.” 258
259
Results 260
Trehalose metabolism genes are overrepresented in dormant spores 261
To determine changes in gene expression that occur during spore germination into 262
yeast, total RNA was isolated from spores undergoing synchronous germination for whole 263
genome transcript analysis. Transcripts were competitively hybridized to a whole genome 264
microarray, and the resulting changes in transcript levels were assessed. Co-expressed groups 265
of genes were submitted to the Database for Annotation, Visualization, and Integrated 266
Discovery (DAVID) to assess functional groups and to the Kyoto Encyclopedia of Genes and 267
Genomes (KEGG) to determine placement within pathways. 268
One of the most enriched pathways within dormant spores was the KEGG annotated 269
pathway for “starch and sucrose metabolism." Transcripts of 14 genes in this pathway showed a 270
pattern of regulation in which their transcripts were overrepresented in dormant spores and 271
became rapidly underrepresented during the first two hours of germination (Figure 1A). Some 272
spore-enriched genes that fall into this metabolic pathway have been implicated previously in 273
spore- or germination-specific functions in other systems: SGA1, which is highly similar to a 274
spore-specific glucoamylase in S. cerevisiae is responsible for glycogen degradation during 275
sporulation; SPR1 is a sporulation-specific exo--1,3-glucan hydrolase in S. cerevisiae; and 276
CELA is a germination-specific spore-coat modifying enzyme from the spore-forming amoeba 277
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Dictyostelium discoideum (46–48). Detection of these transcripts suggests that their roles may 278
be conserved across phyla. In addition, nine of the spore-enriched transcripts in this pathway 279
are involved directly in the biosynthesis and breakdown of trehalose or glycogen (Figure 1A, 280
1B). 281
Among these genes was CNG00690, predicted to encode a trehalose-degrading 282
enzyme. The presence of CNG00690 was unexpected because previous analyses of trehalose 283
synthesis and degradation in C. neoformans had not identified this gene (24, 25). Trehalose 284
biosynthesis genes had been shown to be critical for both the initiation of sexual development 285
(TPS1) and virulence in animal models (TPS1 and TPS2), but deletion of the only known neutral 286
trehalase, NTH1, yielded only a modest decrease in growth on trehalose and no discernable 287
phenotypes in sexual development or virulence (24). This finding was somewhat surprising 288
because many fungi harbor more than one trehalase gene, and their products often play key 289
roles in fungal metabolism (49–51). Our discovery of the enrichment of CNG00690 transcript in 290
dormant spores led us to hypothesize that this gene (which we have named NTH2 for Neutral 291
TreHalase 2) could encode an uncharacterized trehalase with specialized functions in sexual 292
development and/or spore biology. 293
The presence of NTH2 is consistent with the fact that fungi often harbor both cytosolic 294
and secreted trehalases with characteristic features: cytosolic trehalases usually contain a 295
calcium binding domain and a single trehalase domain; secreted trehalases harbor a secretion 296
signal and two trehalase domains (52, 53). Through their predicted functional domains and 297
features, NTH1 and NTH2 appear to be the only two genes encoding trehalase activity in the C. 298
neoformans genome; NTH1 is a predicted cytosolic trehalase, and NTH2 is a predicted secreted 299
trehalase (Figure 1C). The presence of NTH2 may account for the lack of phenotypes in NTH1 300
deletion strains in previous studies and opens the possibility that the degradation of trehalose 301
could play key roles in C. neoformans biology. 302
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NTH1 and NTH2 are differentially regulated under sexual development conditions 304
To determine the expression profiles of NTH1 and NTH2 under different conditions, we 305
carried out RNA blot hybridization using RNA from wild type strains grown in rich medium (YPD 306
broth, mid-log phase), from a cross (a x on V8 pH 7.0 for 72 hours), and from purified spores. 307
As suspected based on previous reports, NTH1 transcript was abundant in cells grown in rich 308
medium, cross conditions, and in spores, suggesting that this trehalase is expressed 309
constitutively (25). In contrast, expression of NTH2 was differentially regulated. While NTH2 310
transcript was abundant in both crosses and purified spores, none was detected from cells 311
grown in rich medium (Figure 2A). 312
To determine the extent to which NTH2 expression was dependent on signals from V8 313
agar medium vs. the process of sexual development, we analyzed RNA from yeast propagated 314
on V8 agar in the absence or presence of a mating partner for 48 hours. NTH2 transcript levels 315
increased on V8 medium both in the absence and presence of a mating partner. This finding 316
indicates that NTH2 is induced largely by V8 medium alone (the same condition that promotes 317
sexual development). (Figure 2B). In contrast to the constitutive expression of NTH1, NTH2 318
expression is responsive to changes in metabolic conditions. 319
320
NTH1 and NTH2 are required for trehalose metabolism 321
To determine the functions of NTH1 and NTH2, we created single and double deletion 322
strains and evaluated them for discernable phenotypes, including the ability to use trehalose as 323
a sole carbon source. Wild type and mutant strains (nth1∆, nth2∆, and nth1∆nth2∆) were grown 324
on solid synthetic medium containing 2% glucose or 2% trehalose as the sole carbon source 325
(Figure 3A). Wild type and all of the trehalase deletion strains (nth1, nth2, and nth1 nth2) 326
grew similarly on medium containing glucose. However, on medium containing trehalose as the 327
sole carbon source, nth1 strains grew more slowly and to a lower density than 328
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wild type strains, nth2 strains showed no difference in growth compared to wild type, and 329
nth1∆ nth2∆ double mutants showed severe growth defects with little or no growth on trehalose-330
containing medium. Together with the observation that NTH1 and NTH2 contain the only 331
identifiable trehalase domains in the C. neoformans genome, these results indicate that both 332
NTH1 and NTH2 encode functional trehalases and provide compelling evidence that these are 333
the only trehalases in C. neoformans. Furthermore, given that NTH2 can partially compensate 334
for the loss of NTH1, these trehalases harbor overlapping (redundant) functions in trehalose 335
catabolism in vegetatively growing cells. 336
Given this relationship during vegetative growth, we hypothesized that the contributions 337
of NTH1 and NTH2 during sexual development would also be at least partially redundant. In the 338
absence of NTH1 and NTH2, we predicted that trehalose would accumulate and that this 339
accumulation would be additive in the double mutant. To test this hypothesis trehalose levels 340
were quantified from wild type and mutant crosses after 5 days on V8 medium. In contrast to our 341
expectations, we observed that deletion of NTH1 and NTH2 had opposing effects on trehalose 342
accumulation: nth1 crosses accumulated significantly more trehalose than wild type crosses, 343
whereas nth2 crosses harbored significantly less trehalose than wild type (p < 0.05 in both 344
cases). The double mutant crosses (nth1 nth2) produced levels of trehalose similar to wild 345
type (Figure 3B). These data indicate a complex relationship between NTH1 and NTH2 in 346
trehalose homeostasis during development that may be due (at least in part) to the predicted 347
differences in localization between Nth1 and Nth2 (Nth1 cytoplasmic and Nth2 secreted). The 348
levels of intracellular trehalose and glucose are likely influenced by both external and internal 349
sources, and the activities of Nth1 and Nth2 on different pools of trehalose could have 350
significant effects on the outcome of overall trehalose levels. It is also possible that differences 351
in localization of trehalase activity among specific cell types (e.g. yeast vs. basidia) that control 352
local trehalose levels were not detected using this assay due to homogenization of trehalose 353
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levels across the entire population of developing cells (yeast, filaments, basidia, and spores). 354
355
Deletion of NTH1 and NTH2 leads to a defect in sporulation 356
To investigate the role of trehalases in sexual development, nth1∆, nth2∆, and 357
nth1∆nth2∆ mutant strains were crossed with wild type strains and with each other under sexual 358
development conditions. Crosses between wild type strains and all of the mutants (nth1∆, 359
nth2∆, and nth1∆nth2∆) were indistinguishable from wild type a x crosses as were nth1∆ x 360
nth1∆ and nth2∆ x nth2∆ crosses (Figure 4). In stark contrast, however, crosses between 361
nth1nth2 mutants (nth1nth2 x nth1nth2) led to severe defects in spore formation, 362
indicating that both genes have redundant functions in this process. 363
At low microscopic resolution, nth1nth2 x nth1nth2 crosses were observed to be 364
similar to wild type crosses with abundant filaments radiating from the periphery of cells 365
undergoing sexual development (Figure 4A). However, under careful examination at higher 366
magnification it is clear that the majority of basidia in nth1nth2 crosses did not develop spore 367
chains, and strikingly, these sporeless basidia appeared extremely enlarged compared to wild 368
type basidia (Figure 4B). In addition, nth1nth2 crosses exhibited the production of an 369
unusually high number of large, ball-like structures, giving the developing filaments a "beads-on-370
a-string" appearance (Figure 4C). Structures like these have been described previously and 371
referred to as chlamydospores (26). The purpose of this cell type is poorly understood; however, 372
they have been observed to be metabolically active, to harbor glycogen, and to be capable of 373
budding off new yeast. It has been hypothesized that C. neoformans chlamydospores are an 374
alternative fate for sexually developing cells and a means of producing new satellite colonies 375
independent of sporulation (26). The structures produced in abundance by nth1∆nth2∆ crosses 376
were also capable of budding off yeast, were found to harbor glycogen via iodine staining (data 377
not shown), and were morphologically indistinguishable from the previous description of 378
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chlamydospores. These features led us to conclude that the nth1∆nth2∆ structures were very 379
likely to be the same type of cells as those previously designated as chlamydospores in C. 380
neoformans. (26). Given the phenotypes of nth1∆nth2∆ mutant strains, we posit that the ratio of 381
trehalose to glucose in basidia is a determinant of cell fate: The cleavage of trehalose by NTH1 382
and NTH2 drives spore formation, whereas the accumulation of trehalose in the nth1∆nth2∆ 383
strain shifts the developmental path, resulting in a marked increase in an alternate cell type 384
(chlamydospore) production. 385
386
Spores harboring trehalase deletions show defects in germination 387
Given the enrichment of NTH2 transcripts in spores and the importance of trehalase 388
function in sporulation, we hypothesized that trehalases could also be important for germination. 389
Despite the observation that nth1 nth2 x nth1 nth2∆ crosses show a marked decrease in 390
spore numbers, these crosses still produced a limited number of spores that were 391
morphologically indistinguishable from wild type spores by light microscopy (data not shown). 392
To test whether spores from trehalase-deficient crosses were viable, spores were isolated from 393
wild type, nth1, nth2, and nth1 nth2 crosses, counted, and grown on rich medium. As 394
expected, nearly 100% of wild type spores germinated and grew into colonies (Figure 5). 395
Similarly, spores from nth1∆ and nth2∆ crosses also germinated at wild type frequencies. In 396
contrast, however, spores from nth1nth2 crosses showed significant decreases in 397
germination frequency, with only 33% of spores forming colonies (Figure 5). These data indicate 398
that not only do nth1 nth2 crosses lead to aberrant basidium formation and a decrease in 399
spore formation, but they also result in substantial numbers of spores that are unable to grow in 400
response to germination conditions. These findings again indicate overlapping functions of 401
NTH1 and NTH2. 402
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The nth2∆ deletion strain is hypervirulent in a mouse model of cryptococcosis 404
To evaluate the roles of trehalase function in pathogenesis, we assessed virulence of 405
the trehalase mutant strains in a mouse tail vein model of infection. It had been shown 406
previously that deletion of NTH1 alone had no effect on virulence (25). This result was initially 407
surprising because of the predicted important roles for trehalose in stress tolerance and the 408
apparent absence of other trehalases in the genome (17). However, our identification of NTH2, 409
and the obviously redundant roles of these genes described above, raised the possibility that 410
deletion of NTH1 resulted in no phenotype due to the presence of NTH2-derived trehalase 411
activity (Figure 3A). 412
Wild type, nth1, nth2, or nth1 nth2 strains were inoculated into mice via tail vein at 413
1x106 yeast cells/mouse. As expected, the effects of wild type and nth1∆ strains were 414
indistinguishable from one another, causing mean times to death of 34 and 35 days respectively 415
(Figure 6). In contrast, however, the nth2 and nth1 nth2 strains both caused morbidity in 416
mice significantly faster (p<0.05)(average time to death 27 and 28 days respectively) than wild 417
type and nth1∆ strains, indicating increased virulence in the absence of nth2∆. This result is 418
consistent with the previous findings that strains harboring deletions of the trehalose 419
biosynthesis genes TPS1 and TPS2 are less virulent. It seems likely that deletion of the 420
biosynthesis genes would decrease the abundance of trehalose, while deletion of the NTH2 421
trehalase would increase overall abundance. Together, these results would support the 422
hypothesis that the level of trehalose, which has been linked to environmental stress resistance 423
in other organisms, may be important for survival against stressors in the host as well. 424
To test this hypothesis, we evaluated the ability of the strains to resist heat shock (at 425
temperatures 37°C, 42°C, and 50°C) and survive exposure to reactive oxygen species (in an 426
H2O2 disk diffusion assay). We also tested capsule inducing conditions (RPMI + 5% CO2 at 427
37°C), melanizing medium (L-DOPA agar), and cell wall stressing agents (1.5 mg/mL calcofluor 428
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white, 0.5 mg/mL caffeine) (54). In no case did the nth1, nth2, or nth1 nth2 mutant strains 429
respond differently than the wild type strain (data not shown). The trehalase mutants were not 430
intrinsically more resistant to global stressors than their wild type counterparts, suggesting that 431
changes in trehalose levels alone were not responsible for the hypervirulence of nth2∆ strains. 432
A surprising aspect of the virulence data was that that the functions of NTH1 and NTH2 433
were not overlapping. In the absence of NTH2 alone, there was a hypervirulence phenotype, 434
indicating that NTH1 was not compensating for the loss of NTH2 activity. Furthermore, there 435
was no change in the phenotype in absence of both NTH1 and NTH2. While the NTH1 and 436
NTH2 activities were largely redundant outside of the host, it appeared that their functions 437
diverge within the host, implicating an NTH2-specific function or feature in modulating virulence. 438
439
Discussion 440
Using transcriptional profiling, we discovered that spores of the human fungal pathogen 441
C. neoformans contain high levels of transcripts associated with the synthesis and breakdown of 442
the disaccharide trehalose, including a previously unrecognized trehalase, NTH2. Through 443
molecular and genetic analyses we revealed that NTH2 and a previously characterized 444
trehalase, NTH1, are fully responsible for trehalose utilization in C. neoformans and have largely 445
overlapping trehalase functions, even though Nth1 is predicted to be cytosolic and Nth2 446
secreted. Significantly, we show for the first time that trehalose degradation is a key function in 447
C. neoformans important for fruiting body formation, spore biogenesis, and germination. 448
Trehalases play a critical role in signaling cell fate decisions: In the absence of trehalases very 449
few spores are produced during sexual development, and instead, development shifts to the 450
formation of chlamydospores. The phenotypes of NTH1 and NTH2 diverge in a mouse model of 451
infection where the deletion of NTH2 results in hypervirulence. 452
453
Trehalose homeostasis determines cell fate during sexual development 454
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Trehalose has been shown to play two primary roles within microbes: First, trehalose is 455
a reserve carbohydrate that can be accumulated and cleaved into two glucose molecules when 456
external carbon sources are scarce, and second, trehalose acts as an osmostabilizer and stress 457
protectant (17). In C. neoformans trehalose biosynthesis is required for initiating sexual 458
development, but roles for trehalose degradation in sexual development have not been 459
documented in any system (23, 55). Our data now bring the full trehalose pathway to light and 460
show that trehalose degradation influences many aspects of C. neoformans biology and is 461
critical for complete sexual development. Notably, in the absence of trehalase genes, 462
development shifts from the formation of fruiting bodies and spores to the formation of what 463
appears to be an alternate cell type known as chlamydospores (26). While little is known about 464
the properties of chlamydospores in C. neoformans, they have been postulated to be structures 465
from which yeast can bud off and establish new colonies (26). Lin and Heitman propose that 466
nutrient sensing through the accumulation of carbohydrate storage molecules, particularly 467
glycogen, may drive cell fate decisions between chlamydospore formation and sporulation. 468
However, glycogen biosynthesis genes, whose transcripts are also highly enriched in spores, 469
are dispensable for both chlamydospore formation and sporulation (26), implicating signaling 470
roles for other carbohydrates. Our findings indicate that trehalose functions as a metabolic 471
signal for determining whether filamentous growth should be terminated with spore production, 472
or whether conditions are sufficiently favorable to continue proliferating (via chlamydospores) 473
and return to vegetative growth. 474
Based on these findings, we propose a model in which trehalose levels are a readout for 475
glucose availability (refer to Figure 1B). In this model as glucose becomes limiting, sexual 476
development can initiate, and spore formation is favored; trehalose levels remain low because 477
of limited substrates for its production, indicating the metabolic need to differentiate into spores. 478
Spores are the terminal consequence of development under these conditions, and the current 479
colony is thus abandoned by dispersed spores for growth in a more favorable environment 480
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elsewhere. However, if nutrient stores are replenished at any point during filamentation, 481
trehalose levels rise (as glucose-derived substrates become plentiful), indicating a change in 482
nutrient availability, and normal growth and colony expansion can be restored through 483
chlamydospore production. 484
This model is consistent with the seemingly paradoxical result that deletion of trehalose 485
synthase (TPS1) and deletion of the trehalases (NTH1 and NTH2) both result in severe defects 486
in sporulation. In both cases the substrates derived from glucose to make trehalose (i.e. 487
glucose-6-phosphate and UDP-glucose) are not used (as in tps1∆) or needed (as in 488
nth1∆nth2∆), resulting in a change in metabolic flux toward carbohydrate storage as glycogen. 489
Given that chlamydospores are hypothesized carbohydrate storage structures, a shift toward 490
glycogen storage could result in a shift in development. In this way trehalose concentration acts 491
as a sensor of metabolic conditions to toggle between two different developmental pathways, 492
thus controlling a critical cell fate decision by playing a novel signaling role, rather than simply 493
functioning only as a fuel source or osmoprotectant. This is akin to the role of trehalose 494
signaling in plants that serves to regulate development and plant physiology. However, plants 495
do not accumulate trehalose to the high levels typical of fungi, and thus, the contribution of 496
trehalose metabolism to signaling vs. stress responses and nutrition is not well understood in 497
plants. The increased development of chlamydospores in trehalase deficient C. neoformans 498
may serve as a platform to dissect the different contributions of trehalose metabolism on the 499
physiology of fungal pathogens. 500
501
Metabolic adaptation, differentiation, and consequences for virulence 502
In this work, we discovered that trehalose degradation affects spore germination, which 503
is critical for the long-term survival of the organism in both the natural environment and a host 504
lung. In the absence of NTH1 and NTH2 spore germination drops three-fold, suggesting that 505
trehalase function could be important for stabilizing dormant spores, cleaving trehalose into 506
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glucose for fuel during germination, or both. The fact that both rich media conditions and the 507
mammalian lung are germination-inducing (3, 11, 12) underscores a characteristic feature of C. 508
neoformans - the ability to survive and grow in rapidly changing, environmental conditions. The 509
tissues of a mammalian host are an unusual environment for C. neoformans to inhabit, and 510
thus, the ability to respond to these conditions is likely shaped by the selection pressures of its 511
typical environmental niches (56, 57). The advantages potentially conferred by this relationship 512
have been proposed to explain many of the survival behaviors exhibited by C. neoformans in 513
the host. For example, Steenbergen et al. posit that the unusual ability of C. neoformans to 514
survive and grow within macrophages may result from adaptive strategies developed in the 515
natural environment in response to soil amoeba (58). 516
In the same vein, it would seem likely that adaptations to efficiently access internal 517
stores of energy (e.g. trehalase activity to cleave accumulated trehalose) and scavenge 518
trehalose from the environment would enhance survival in the host. Surprisingly, however, we 519
discovered that an nth2∆ strain is hypervirulent in a mouse model of infection, indicating a 520
negative correlation between trehalase function and virulence in the mouse host. That is, the 521
presence of an intact trehalose-degrading pathway confers a disadvantage in disease-causing 522
ability. On possible explanation for this finding may lie in the fact that Nth2 is predicted to be 523
secreted. Perhaps Nth2 is an antigen that can be recognized by the adaptive immune system, 524
allowing the host immune response more opportunity to inhibit C. neoformans growth. 525
Altering trehalose metabolism in other fungal pathogens has been shown to both 526
positively and negatively affect virulence in murine models of infection. Broadly, trehalose 527
accumulation has been associated with increased stress-resistance that typically results in 528
increased infectivity or virulence (17). However, changes in trehalose accumulation can result in 529
changes in cell wall structure and morphogenesis that may also affect the ability of fungal 530
pathogens to cause disease (20, 21, 23). There may be changes that occur in trehalase 531
deficient C. neoformans yeast inside of a mammalian host, such as cell wall polysaccharide 532
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composition or titan cell formation (enlarged yeast cells associated with virulence), that can be 533
elucidated in future studies of the interaction of trehalase deficient C. neoformans and the host. 534
535
Intersections among sexual development, metabolism, and pathogenesis 536
Spores as infectious particles form an obvious connection between the processes of 537
sexual development and mammalian pathogenesis, but the full relationship is more complex. In 538
this work, we reveal a link between the processes of spore production and virulence in a 539
mammalian host through the actions of trehalose-degrading enzymes. This unexpected 540
connection underscores the emerging relationships between sexual development and virulence 541
among human fungal pathogens in general and the role of metabolic acclimation in C. 542
neoformans in particular. Responses in C. neoformans to the extreme conditions between 543
natural environment and human host are of particular interest because understanding metabolic 544
adaptations promises to reveal pathways that influence pathogenesis. 545
The importance of these relationships is underscored by recent studies of C. 546
neoformans morphology in which filamentous growth of C. neoformans results in a loss of 547
virulence in mice, implying that the yeast morphology plays a role in pathogenesis. A coincident 548
possibility, however, is that morphology is secondary to changes in metabolic gene expression 549
between yeast responding to environmental signals (such as those that initiate developmental 550
changes) and yeast responding to host conditions. The resulting metabolic acclimation results in 551
both positive and negative effects on pathogen survival in the host and could help to explain 552
differences in virulence properties observed between clinical and environmental isolates. One 553
possibility is that gene expression patterns that would normally be advantageous in an 554
environmental niche but disadvantageous within a host are modified, thus making the host a 555
selective environment for cells that have the capacity to rapidly modulate expression of 556
metabolic or stress response pathways. Discovering these pathways provides another route 557
toward understanding how host resources may be enhanced to prevent fungal infection and/or 558
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disease. 559
560
Acknowledgements 561
Thank you to A. Adams, L. Ekena, S. Peters, and J. Kusiak for laboratory support and N. Walsh, 562
M. Mead, and C. Fox for comments on the manuscript. Thank you to the University of Wisconsin 563
Laboratory Animal Resource Staff for the care of animals used in this study. This work was 564
supported by NIH R01 AI064287, NIH R01 AI059370, and a Burroughs Wellcome Fund Career 565
Award in Biomedical Sciences, all to CMH. MRB was supported by the University of Wisconsin 566
Molecular Biosciences Training Grant NIH T3GM07215. 567
568
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Figure Legends 569
Figure 1. Trehalose metabolism gene transcripts are overrepresented in spores. A. Heat 570
map of genes in the KEGG starch and sucrose metabolism pathway that were spore-enriched. 571
Relative fold change is represented from -4 (blue) to +4 (yellow). Each column represents a 572
time point of germination in hours. Dormant spore transcripts are represented in "0." Yeast 573
transcripts are represented in "10." Each row represents a gene. B. A schematic of genes 574
predicted to act in the trehalose and glycogen metabolic pathway with those found to be spore-575
enriched in red. Only one gene, NTH1, was not found to be spore-enriched. C. A schematic of 576
predicted C. neoformans trehalases, Nth1 and Nth2, and their functional domain content, 577
including family 37 glycosyl hydrolase ,-trehalase domain (GH37, Blue) calcium binding 578
regulatory domain (Ca, green), and predicted signal peptide (purple). 579
580
Figure 2. NTH1 and NTH2 are differentially expressed. A. NTH2 is induced during sexual 581
development. Lanes 1 and 2: a and yeast grown in rich medium (YPD liquid), respectively. 582
Lane 3: a and yeast mixed under cross conditions (V8 agar pH 7 at 22°C for 72 hours). Lane 583
4: spores from a cross after 72 hours. Top, middle, and bottom panels reflect probes to the 584
transcripts of interest: NTH2, NTH1, and GPD1, respectively. B. NTH2 is induced on V8 agar. 585
Lanes 1 and 2: a and yeast grown on rich medium (YPD agar), respectively. Lanes 3 and 4: a 586
and yeast grown individually on V8 agar for 48 hours. Lane 5: a and yeast grown in mixed 587
culture on V8 agar for 48 hours. Probes used were to NTH2 and GPD1. GPD1 was used as a 588
loading and hybridization control. 589
590
Figure 3. Deletion of NTH1 and NTH2 causes a severe synthetic defect in trehalose 591
utilization. A. Wild type a, wild type , a nth1∆, nth1∆, a nth2∆, nth2∆, a nth1∆nth2∆ and 592
nth1∆nth2∆ strains (left) were struck onto synthetic defined media containing either 2% glucose 593
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(middle) or 2% trehalose (right) as a sole carbon source and grown for 2 days at 30°C. B. 594
Trehalose was quantified in wild type and mutant crosses after 5 days on V8 pH 7.0. Mean 595
trehalose values from three biological replicates were compared using one-way ANOVA 596
statistics, and those crosses that differed significantly (p < 0.05) from wild type (a x ) are 597
marked with an asterisk. 598
599
Figure 4. Deletion of NTH1 and NTH2 leads to aberrant sexual development. A. Wild type, 600
nth1∆, nth2∆, and nth1 nth2 crosses after five days all show normal filament formation. 100X 601
magnification; bar = 100 µm. B. nth1∆nth2∆ crosses form large, ball-like structures in place of 602
basidia and do not produce spore chains. 400X magnification; Bar = 10 µm. C. After seven days 603
of development nth1 nth2∆ crosses produced numerous large, swollen nodules on filaments 604
giving them beads-on-a-string appearance (black arrows), consistent with previous descriptions 605
of chlamydospores. 200X magnification; bar = 50 µm. 606
607
Figure 5. Trehalase deficient spores are defective for growth. Spores from wild type, nth1, 608
nth2, and nth1nth2 crosses were isolated, counted, and cultured on rich medium for 3 days 609
at 30°C. X-axis represents genotype of spores. Y-axis represents percent of spores plated that 610
produced a colony. Resulting mean germination frequencies for three independent biological 611
replicates were compared using ANOVA, with only nth1Δnth2Δ spores having a significant 612
reduction (p<0.05) in germination. 613
614
Figure 6. Strains harboring nth2∆ are hypervirulent in mice. X-axis shows time in days after 615
tail vein infection with 1 x 106 wild type (diamonds), nth1 (squares), nth2∆ (triangles), or 616
nth1nth2∆ (cross-hatches) yeast. Y-axis shows percent survival of five Balb/c mice per 617
strain. The difference in average time to death between nth2 and nth1nth2 strains vs. wild 618
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type and nth1∆ strains is 7 days and is both statistically significant (Wilcoxon rank-sum test, 619
p<0.05) and reproducible between experiments. 620
621
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Table I. Strains and Oligonucleotides Used in This Study
Strain Genotype Reference
C. neoformans var. neoformans serotype D
JEC20 MATa Kwon-Chung et al, 1992
JEC21 MAT Kwon-Chung et al, 1992
CHY2083-2085 MATa nth2::NEOR This study
CHY2089-2091 MAT nth2::NEOR This study
CHY1705-1707 MATa nth1::NATR This study
CHY1711-1713 MAT nth1::NATR This study
CHY2182-2183, 2188 MATa nth1::NATR
nth2::NEOR This study
CHY2180-2181, 2184 MAT nth1::NATR
nth2::NEOR This study
Oligos used in cloning and PCR
CHO1565 TAATCAGTGGGTGAGGTGTG NTH1 5' Flank FWD
CHO1566 GCTCACATCCTCGCAGCCTCGAAGTTGGAGTGAAGGAGA NTH1 5' Flank REV
CHO1567 CTAGTTTCTACATCTCTTCCCTGGAAACATTCTTGATGG NTH1 3' Flank FWD
CHO1568 TCTCGCCCCTCCTAAACTAT NTH1 3' Flank REV
CHO1569 TCTCCTTCACTCCAACTTCGAGGCTGCGAGGATGTGAGC NATR cassette
FWD
CHO1570 CCATCAAGAATGTTTCCAGGGAAGAGATGTAGAAACTAG NATR cassette
REV
CHO2636 GTGTATCCAATCCAAGCACT NTH2 5' Flank FWD
CHO2637 GTCATAGCTGTTTCCTGGAGCTTTCGTTCATCAAGTC NTH2 5' Flank REV
CHO2764 CATCCTCGCAGCAAGGGCGTTTTGAGGTTCGAGGGTGAG NTH2 3' Flank FWD
CHO2765 CCCATACTACCGCAGCAATC NTH2 3' Flank REV
CHO2640 GACTTGATGAACGAAAGCTCCAGGAAACAGCTATGAC NEOR
cassette FWD
CHO2641 GGCTATGTTCAATGGGTATCCGCCCTTGCTGCGAGGATG NEOR
cassette REV
CHO2979 GCCTTCTCGAGGGGCTGGGA NTH1 northern probe FWD
CHO2980 CGTGGGCTTCGCCAGTCGTT NTH1 northern probe REV
CHO2938 GCCGCCCCATGCGTGTAAGT NTH2 northern probe FWD
CHO2939 CTCACCACCTCCTCCAGCGG NTH2 northern probe REV
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