key: cord-0276043-dd6bp661 authors: Jakab, Ágnes; Balla, Noémi; Ragyák, Ágota; Nagy, Fruzsina; Kovács, Fruzsina; Sajtos, Zsófi; Borman, Andrew M.; Pócsi, István; Baranyai, Edina; Majoros, László; Kovács, Renátó title: Transcriptional profiling of the Candida auris response to exogenous farnesol exposure date: 2021-08-24 journal: bioRxiv DOI: 10.1101/2021.08.23.457447 sha: 95318572e1c5467dd7cb281215ac598813e6e379 doc_id: 276043 cord_uid: dd6bp661 The antifungal resistance threat posed by Candida auris necessitates bold and innovative therapeutic options. Farnesol, a quorum-sensing molecule with a potential antifungal and/or adjuvant effect; it may be a promising candidate in alternative treatment regimens. To gain further insights into the farnesol-related effect on C. auris, genome-wide gene expression analysis was performed using RNA-Seq. Farnesol exposure resulted in 1,766 differentially expressed genes. Of these, 447 and 304 genes with at least 1.5-fold increase or decrease in expression, respectively, were selected for further investigation. Genes involved in morphogenesis, biofilm events (maturation and dispersion), gluconeogenesis, iron metabolism, and regulation of RNA biosynthesis showed down-regulation, whereas those related to antioxidative defense, transmembrane transport, glyoxylate cycle, fatty acid β-oxidation, and peroxisome processes were up-regulated. In addition, farnesol treatment increased the expression of certain efflux pump genes, including MDR1, CDR1, and CDR2. Growth, measured by change in CFU number, was significantly inhibited within 2 hours of the addition of farnesol (5.8×107±1.1×107 and 1.1×107±0.3×107 CFU/ml for untreated control and farnesol-exposed cells, respectively) (p<0.001). In addition, farnesol treatment caused a significant reduction in intracellular iron (152.2±21.1 vs. 116.0±10.0 mg/kg), manganese (67.9±5.1 vs. 18.6±1.8 mg/kg), and zinc (787.8±22.2 vs. 245.8±34.4 mg/kg) (p<0.05–0.001) compared to untreated control cells, whereas the level of cooper was significantly increased (274.6±15.7 vs. 828.8±106.4 mg/kg) (p<0.001). Our data demonstrate that farnesol significantly influences the growth, intracellular metal ion contents, and gene expression related to fatty acid metabolism, which could open new directions in developing alternative therapies against C. auris. Importance Candida auris is a dangerous fungal pathogen that causes outbreaks in health care facilities, with infections associated with high mortality rate. As conventional antifungal drugs have limited effects against the majority of clinical isolates, new and innovative therapies are urgently needed. Farnesol is a key regulator molecule of fungal morphogenesis, inducing phenotypic adaptations and influencing biofilm formation as well as virulence. Alongside these physiological modulations, it has a potent antifungal effect alone or in combination with traditional antifungals, especially at supraphysiological concentrations. However, our knowledge about the mechanisms underlying this antifungal effect against C. auris is limited. This study has demonstrated that farnesol enhances the oxidative stress and reduces the fungal survival strategies. Furthermore, it inhibits manganese, zinc transport, and iron metabolism as well as increases fungal intracellular copper content. In addition, metabolism was modulated towards β-oxidation. These results provide definitive explanations for the observed antifungal effects. Gene set enrichment analyses on the up-regulated and down-regulated gene sets were 169 performed with Candida Genome Database Gene Ontology Term Finder 170 (http://www.candidagenome.org/cgi-bin/GO/goTermFinder), using function, process, and 171 component gene ontology (GO) terms. Only hits with a p value of < 0.05 were considered in 172 the evaluation process (Table S2) . 173 Besides GO terms, groups of functionally related genes were also generated by extracting 174 data from the Candida Genome Database (http://www.candidagenome.org) unless 175 otherwise indicated. The enrichment of C. auris genes from these gene groups in the up-176 regulated and down-regulated gene sets was tested with Fisher's exact test (p < 0.05). The 177 following gene groups were created: 178 (i) Virulence-related genes -Genes involved in the genetic control of C. albicans virulence 179 were collected according to Mayer et al. (2013) , Höfs et al. (2016) , and Araújo et al. (2017) 180 (15-17) . (iv) Iron metabolism-related genes -Genes involved in iron acquisition by C. albicans were 189 collected according to Fourie et al. (2018) (18). 190 (v) Zinc, manganese, and copper homeostasis genes -Genes involved in zinc and copper 191 acquisition were collected according to Gerwien et al. (2018) (19) . 192 The complete gene lists of the above-mentioned gene groups are available in Supplementary 193 Table 3 . 194 195 Assays of iron, manganese, zinc, and copper contents of C. auris cells 196 C. auris pre-cultures were grown, and farnesol exposure was performed as described above. 197 samples were calculated and expressed in DCM units (mg/kg), as described previously by 204 Jakab et al. 2021 (20) . The metal contents of the biomasses were determined in triplicate, 205 and mean ± SD values were calculated. Statistical significance of changes was determined by 206 two-way ANOVA. Significance was defined as a p value of < 0.05. 207 208 209 The growth of C. auris was examined following 75 µM farnesol treatment in YPD. Adding 212 farnesol to pre-incubated cells resulted in a remarkable growth inhibition, starting at 6 hours 213 post inoculation, which was confirmed by both absorbance (OD 640 ) measurements and CFU 214 determination. Growth was significantly inhibited within 2 hours of the addition of farnesol 215 as assessed both by CFU changes (5.8 × 10 7 ± 1.1 × 10 7 and 1.1 × 10 7 ± 0.3 × 10 7 CFU/ml for 216 untreated control and farnesol-exposed cells, respectively) (p < 0.001) and observed 217 absorbance values (1.28 ± 0.04 and 0.72 ± 0.04 for untreated control and farnesol-exposed 218 cells, respectively, at OD 640 nm) (p < 0.001) (Fig. 1) . The observed growth inhibition was 219 further confirmed by changes in measured DCM at 12 hours incubation time (5.5 ± 0.2 and 220 1.3 ± 0.1 g/L for untreated control and farnesol-exposed cells, respectively) (p < 0.001). Comparison of the farnesol-exposed C. auris global gene expression profile with that of 232 unexposed cells revealed 1,766 differentially expressed genes. Among those, 447 were up-233 regulated and 304 were down-regulated in the farnesol-exposed samples compared to the 234 untreated controls (Figs. 3 and 4, Tables S2 and 3) . 235 236 To identify larger patterns in differential gene expression and to obtain an overall insight 238 into the impact of farnesol, gene ontology terms were assigned to all of the genes in the C. 239 auris genome; afterwards, we compared the terms for both the down-regulated and up-240 regulated genes to a background of all terms. We found 19 and 22 significant gene groups 241 that were underrepresented and overrepresented in this analysis, respectively ( Virulence-related genes were significantly enriched within the farnesol-responsive down-246 regulated gene group, according to Fisher's exact test (Table S3) . 247 Most of these 11 putative genes are involved in biofilm maturation (RBT1, HWP1, BCR1, 248 EFG1, DSE1, BRG1, UME6, ZAP1, and RLM1) and dispersion (NRG1, and UME6) (Figs. 3 and 4, 249 Table S3 ); also, five down-regulated morphogenesis genes (EFG1, HWP1, HGC1, WAL1, and 250 VRP1) are notable (Fig. 3 , Table S3 ). Down-regulation of RBT1 and NRG1 under farnesol 251 treatment was also supported by RT-qPCR data ( Fig. 5 and Table S4) . Table S3 ). In addition, farnesol exposure increased the expression of 258 HSP21, YPD1, and HOG1, encoding small heat shock protein, phosphorelay protein, and MAP 259 kinase (Fig. 3 , Table S3 ). Upregulation of CAT1 and CCP1, coding for catalase and 260 cytochrome-c peroxidase in farnesol-treated cells, was also confirmed by RT-qPCR ( Fig. 5 and 261 Table S4 ). 262 263 Selected genes involved in glucose catabolism and fatty acid metabolism were determined 265 with the Candida Genome Database (http://www.candidagenome.org). Farnesol treatment 266 down-regulated PCK1 and FBP1, encoding key enzymes specific to gluconeogenesis, but not 267 glycolysis and tricarboxylic acid cycle genes (Fig. 3 , Table S3 ). In addition, three genes related 268 to the glyoxylate cycle (ACO2, ICL1, and MDH1-3) were significantly enriched in the up-269 regulated gene set (Fig. 3 , Table S3 ). Table S3 ). 279 The up-regulation of POT1 (3-oxoacyl CoA thiolase) and the down-regulation of INO1 and 280 FTR1 were supported by RT-qPCR data ( Fig. 5 and Table S4) . 281 Farnesol treatment led to the increased expression of numerous genes (60 genes altogether) 284 involved in transmembrane transport, including 5 putative antifungal drug transporter genes 285 (MDR1, CDR1, CDR4, HOL3, and YOR1), 4 putative carbohydrate transport genes (HGT2, 286 HGT17, HGT19, and HXT5), 13 putative amino acid transport genes, as well as 4 putative 287 phosphate and sulfate transporter genes (PHO84, PHO89, GIT1, and SUL2) (Figs. 3 and 4, 288 Table S3 ). Farnesol exposure also caused a significant increase in the expression of CDR1 and 289 MDR1 (ABC transporters) as well as HGT2 (glucose transmembrane transporter) of treated 290 cells, according to the RT-qPCR results ( Fig. 5 and Table S4 ). 291 292 Farnesol treatment caused a significant reduction in intracellular iron, manganese, and zinc 294 content (p < 0.05-0.001) compared to untreated control cells, whereas the level of 295 intracellular copper was significantly increased, as shown in Table 1 Alternative treatments interfering with quorum-sensing have recently become attractive 299 therapeutic strategies, particularly against difficult-to-treat multidrug-resistant pathogens 300 such as C. auris (9, (21) (22) . Previous studies have reported that fungal quorum-sensing 301 molecules may have a remarkable antifungal effect and/or a potent adjuvant effect in 302 combination with traditional antifungal agents (7,10,23-26). For example, Nagy et al. (2020) 303 reported that supraphysiological farnesol exposure caused a significant reduction in the 304 growth rate and metabolic activity of C. auris planktonic cells and biofilms, respectively (7). 305 In addition, 75 μM farnesol treatment significantly decreased the fungal kidney burden in an 306 immunocompromised systemic mouse model (7). Total transcriptome analysis using RNA-307 Seq may be an important technique to fully understand the underlying mechanisms of the 308 observed antifungal effect exerted by these molecules. 2020) (7). In this study, several putative oxidative stress-responsive genes, namely CAT1 313 (encoding for catalase activity), GPX1 (encoding glutathione peroxidase), and SOD1, SOD2, 314 SOD6 (encoding superoxide dismutases), were up-regulated following exposure to farnesol. 315 It is noteworthy that farnesol exposure also up-regulated HOG1 MAP kinase, which is a 316 critical component of the fungal oxidative stress response, further supporting the farnesol-317 induced oxidative stress in C. auris (29) . This fact is further confirmed by the elevated DCF 318 and superoxide dismutase levels in farnesol-exposed cultures (7). 319 Recent transcriptomic data have demonstrated that farnesol treatment affected the 320 transcription of iron homeostasis-related genes, as well as the iron, zinc, manganese, and 321 copper contents of C. auris. The down-regulation of iron uptake genes was associated with 322 the significantly decreased iron content measured in farnesol-exposed cells. Similarly, the 323 menadion sodium bisulphite induced oxidative stress also affected the transcription of iron 324 homeostasis-related genes and the iron content of C. albicans cells (20). It should be noted 325 that this response related to iron decrease may be a part of a general defense mechanism 326 against farnesol and menadion sodium bisulphite to minimize the damage caused by ferrous 327 ions. According to previous studies, elevated free intracellular iron levels facilitate the 328 formation of reactive oxygen species and mediate iron-dependent cell death in 329 Saccharomyces cerevisiae (20,30). 330 The down-regulated expression of CSR1, encoding a major transcription factor that stabilizes 331 zinc homeostasis and provides cells with zinc-dependent protection against farnesol-induced 332 oxidative stress (19), is related to the decreased intracellular zinc level observed. Zinc is an 333 essential transition metal in oxidative stress defense because it is a structural component of 334 superoxide dismutase, which is a key enzyme in the neutralization of superoxide radical 335 In contrast to the majority of metals, manganese acts as an anti-oxidant element at high 337 concentrations rather than a reactive oxygen species producer (19). However, farnesol 338 inhibited the expression of SMF1, which is responsible for maintaining the intracellular 339 manganese levels for anti-oxidant actions (19). In addition, the expression of PMR1 (p < 0.05, 340 FC = 1.2) was also inhibited, decreasing the virulence of fungal cells (31). This was associated 341 with our previously published data, where daily farnesol treatment significantly decreased 342 the virulence of C. auris (7). 343 The observed down-regulation of the copper exclusion system (CRP1/CCC2, encoding P-type 344 ATPases) may be associated with the significantly increased copper contents and the 345 remarkable growth inhibition in farnesol-treated cells. Copper regulates a variety of cellular 346 processes in fungal pathogens. When it presents in excess, it is associated with the 347 generation of reactive oxygen species via the Fenton reaction and destroys the iron-sulfur 348 cluster reducing the viability of cells (19, (32) (33) (34) (35) . The elevated free copper levels in the 349 farnesol-exposed cells may contribute to the increased redox imbalance quantified by DCF 350 production (7), which was accompanied by increases in the specific activity of superoxide 351 dismutase (7). Moreover, recent studies have shown that copper efflux pumps may be 352 equally important in fungal defense strategies against phagocytes as for the virulence in C. 353 albicans (19,32-35) . 354 Interestingly, farnesol exposure exerted a significant up-regulation in several fatty acid β-355 oxidation-related genes (POX1, ECI1, FAT1, FAA21, and POT1) . The elimination of 356 unnecessary membrane lipids and the increased usage of fatty acids may provide a higher 357 metabolic flux, needed for the maintenance of membrane fluidity (36). Jabra Rizk et al. 358 (2006) and Rossignol et al. (2007) described that farnesol influences the membrane 359 permeability in non-albicans species as C. dubliniensis and C. parapsilosis (5-6). The elevated 360 fatty acid oxidation activity may explain the membrane-related farnesol effect, which may 361 elucidate the previously observed antifungal effect (7). A further potential explanation of the 362 antifungal effect can be found in the down-regulation of ergosterol biosynthesis-related 363 genes, which alter the membrane permeability and/or fluidity (37) . In our study, the ERG6 364 gene was down-regulated following farnesol exposure, which may enhance the passive 365 diffusion of farnesol across the membrane; furthermore, the decreased Erg6 content may 366 confirm the higher susceptibility of C. auris cells to oxidative stress (37) (38) . Oliveira et al. 367 (2020) showed that the ERG6 mutant Cryptococcus neoformans displays impaired 368 thermotolerance and increased susceptibility to oxidative stress as well as to different 369 antifungal drugs, explaining, for instance, the previously reported synergizing effect with 370 azoles (7). Furthermore, the ERG6 mutant C. neoformans was totally avirulent in an 371 invertebrate model, which may also explain the reduced virulence of C. auris after daily 372 farnesol treatment (7,37). Beside ERG6, INO1 was also down-regulated following farnesol 373 treatment. This gene encodes the inositol-1-phosphate synthase, a key enzyme in the 374 synthesis of inositol for phosphotidylinositol synthesis. The down-regulation of this gene 375 may further explain the synergizing effect of farnesol with azoles against C. auris (7) Multidrug-Resistant Candida auris 425 Critically Ill Coronavirus Disease Patients, India Spread of Carbapenem-Resistant Gram-Negatives and 431 Candida auris during the COVID-19 Pandemic in Critically Ill Patients: One Step Back in 432 Impact of the SARS-CoV-2 Pandemic in Candidaemia Invasive Aspergillosis and Antifungal Consumption in a Tertiary Hospital Quorum sensing in the dimorphic fungus Candida albicans is mediated by 441 farnesol Effect of farnesol on Candida 444 dubliniensis biofilm formation and fluconazole resistance Transcriptional 448 response of Candida parapsilosis following exposure to farnesol In vitro 452 and in vivo Effect of Exogenous Farnesol Exposure Against Candida auris Farnesol and Tyrosol: Secondary Metabolites with a 456 Crucial quorum-sensing Role in Candida Biofilm Development Fungal Quorum-Sensing Molecules: A Review of Their Antifungal 460 Effect against Candida Biofilms Farnesol 463 increases the activity of echinocandins against Candida auris biofilms Isolates of the emerging pathogen Candida auris 467 present in the UK have several geographic origins Deletion of the fungus specific protein phosphatase Z1 exaggerates the oxidative stress 471 response in Candida albicans Physiological and Transcriptional Responses of Candida parapsilosis to Exogenous 475 A reagent for the single-step simultaneous isola-tion of RNA, DNA 478 and proteins from cell and tissue samples Candida albicans pathogenicity mechanisms Interaction of Candida albicans with host cells: virulence 484 factors, host defense, escape strategies, and the microbiota Portrait of Candida Species Biofilm Regulatory 487 Metals in fungal virulence The Negative Effect of Protein Phosphatase Z1 Deletion on the Oxidative Stress Tolerance of Candida albicans Is Synergistic with 498 Exploring anti-quorum sensing and anti-virulence based strategies to fight 502 Candida albicans infections: an in silico approach Combination Therapy Strategy of Quorum Quenching Enzyme and Quorum Sensing 506 Inhibitor in Suppressing Multiple Quorum Sensing Pathways of Effect 510 of caspofungin and micafungin in combination with farnesol against Candida parapsilosis 511 biofilms The in vitro and in vivo 514 efficacy of fluconazole in combination with farnesol against Candida albicans isolates using a 515 murine vulvovaginitis model Erg6 affects membrane composition and virulence of the human fungal pathogen 568 Cryptococcus neoformans Sequencing, 571 disruption, and characterization of the Candida albicans sterol methyltransferase (ERG6) 572 gene: drug susceptibility studies in erg6 mutants cDNA microarray 576 analysis of differential gene expression in Candida albicans biofilm exposed to farnesol Genomic insights into multidrug-resistance, mating and virulence in Candida 581 auris and related emerging species Transcriptome Assembly and Profiling of Candida auris 585 Abrogation of pathogenic attributes in drug resistant Candida 588 auris strains by farnesol Gene Expression Omnibus: NCBI gene expression 591 and hybridization array data repository Down-regulated (A, blue) and up-regulated (B, red) genes were defined as differentially 633 expressed genes (corrected p value of <0.05). The enrichment of these gene groups were 634 identified with the Candida Genome Database Figure 5 Correlation between RT-qPCR and transcriptome data Relative transcription 640 levels were quantified as ΔΔCP = ΔCP control − ΔCP treated , where ΔCP treated = CP tested gene − 641 ΔCP control = CP tested gene − 642 CP reference gene , measured from control cultures. CP values represent the qRT-PCR cycle 643 numbers of crossing points. The ACT1 gene was used as a reference gene. ΔΔCP values 644 significantly (p < 0.05 by Student's t test downregulated genes) are marked in red and blue, respectively. Pearson's correlation 646 coefficient between the RT-qPCR and RNA-Seq values was 0.87. The data set is available in 647 Supplementary Table 1: Oligonucleotide primers used for RT-qPCR analysis Supplementary Table 2: Results of the gene set enrichment analysis Significant shared GO terms (p < 0.05) were determined with the Candida Genome Database 653 Up-and down-regulated genes were defined as differentially expressed genes where 655 log 2 (FC) > 0.585 or log 2 (FC) < −0.585. The FC ratios were calculated from the normalized 656 gene expression values. Biological processes, molecular function and cellular component 657 categories are provided Supplementary Table 3: Transcription data of selected gene groups Part 1: Genes involved in genetic control of Candida auris virulence Part 2: Genes involved in in selected metabolic pathways Part 3: Genes involved in ergosterol and fatty acid metabolism Genes involved in response to oxidative stress Part 5: Genes involved in metals metabolism Part 6: Selected genes involved in regulation of RNA biosynthetic process Selected genes have protein kinase or phosphatase activity Part 8: Selected genes involved in membrane transport The systematic names, gene names and the features (putative molecular function or 669 biological process) of the genes are given according to the Candida Up-and down-regulated gene were defined as differentially expressed genes with < 0.05 672 corrected p value. RNA-Seq data are presented as FC values, where FC is "fold change Up-and down-regulated genes are marked with red and blue colour Results of gene enrichment analysis (Fisher's exact test) are also enclosed to the parts 1-5. 675 "The response of oxidative stress Supplementary Table 4: Results of the RT-qPCR measurements Relative transcription levels were quantified with ΔΔCP = ΔCP control -ΔCP treated, where 680 ΔCP treated = CP tested gene -CP reference gene was measured from treated cultures and ΔCP control = 681 CP tested gene -CP reference gene was measured from control cultures. CP values represent the qRT-682 RT-qPCR data are presented as mean ± SD calculated 683 from three independent measurements, normalised to the ACT1 gene expression and were 684 compared using Student's t-test (p<0.05). Significantly higher or lower than zero ΔΔCP values 685 (up-or down-regulated gene) are marked with red and blue colours The RNA sequencing data discussed have been deposited in NCBI's Gene Expression 419Omnibus (43)