The CgHaa1-Regulon Mediates Response and Tolerance to Acetic Acid Stress in the Human Pathogen Candida glabrata

To thrive in the acidic vaginal tract, Candida glabrata has to cope with high concentrations of acetic acid. The mechanisms underlying C. glabrata tolerance to acetic acid at low pH remain largely uncharacterized. In this work, the essential role of the CgHaa1 transcription factor (encoded by ORF CAGL0L09339g) in the response and tolerance of C. glabrata to acetic acid is demonstrated. Transcriptomic analysis showed that CgHaa1 regulates, directly or indirectly, the expression of about 75% of the genes activated under acetic acid stress. CgHaa1-activated targets are involved in multiple physiological functions including membrane transport, metabolism of carbohydrates and amino acids, regulation of the activity of the plasma membrane H+-ATPase, and adhesion. Under acetic acid stress, CgHaa1 increased the activity and the expression of the CgPma1 proton pump and contributed to increased colonization of vaginal epithelial cells by C. glabrata. CgHAA1, and two identified CgHaa1-activated targets, CgTPO3 and CgHSP30, are herein demonstrated to be determinants of C. glabrata tolerance to acetic acid. The protective effect of CgTpo3 and of CgHaa1 was linked to a role of these proteins in reducing the accumulation of acetic acid inside C. glabrata cells. In response to acetic acid stress, marked differences were found in the regulons controlled by CgHaa1 and by its S. cerevisiae ScHaa1 ortholog, demonstrating a clear divergent evolution of the two regulatory networks. The results gathered in this study significantly advance the understanding of the molecular mechanisms underlying the success of C. glabrata as a vaginal colonizer.


CAGL0G05632g
6,95 1,20E-07 YDL218W Putative protein of unknown function; YDL218W transcription is regulated by Azf1p and induced by starvation and aerobic conditions; expression also induced in cells treated with the mycotoxin patulin CAGL0I10010g 6,82 1,18E-08 BTN2 v-SNARE binding protein that facilitates specific protein retrieval from a late endosome to the Golgi; modulates arginine uptake, possible role in mediating pH homeostasis between the vacuole and plasma membrane H(+)-ATPase CAGL0M01166g 6,44 2,09E-06 THI4 Thiazole synthase, abundant protein involved in the formation of the thiazole moiety of thiamine during thiamine biosynthesis; acts more as a co-substrate rather than an enzyme by providing the sulphur source for thiazole formation; undergoes a single turnover only; required for mitochondrial genome stability in response to DNA damaging agents Low-affinity glucose transporter of the major facilitator superfamily, expression is induced by Hxk2p in the presence of glucose and repressed by Rgt1p when glucose is limiting

UP-REGULATED GENES
Supplementary Table S1 -List of the genes found to be up-or down-regulated in response to acetic acid (30 mM, at pH 4.0) in C. glabrata KUE100 cells. Genes whose expression increased or decreased (above a 2-fold threshold level) in acetic acid (AC)-challenged cells, in comparison with unstressed cells (CTRL), were selected as described in materials and methods and are here listed. The biological function indicated was based on the information available at Candida Genome Database and/or in the information available for the S. cerevisiae orthologue

CAGL0H04279g
MT-IIB 3,65 2,67E-05 -Copper-binding metallothionein, involved in sequestration of metal ions CAGL0F04895g 3,61 4,57E-06 GPH1 Non-essential glycogen phosphorylase required for the mobilization of glycogen, activity is regulated by cyclic AMP-mediated phosphorylation, expression is regulated by stress-response elements and by the HOG MAP kinase pathway CAGL0K12254g 3,60 1,98E-06 VID24 Peripheral membrane protein located at Vid (vacuole import and degradation) vesicles; regulates fructose-1,6-bisphosphatase (FBPase) targeting to the vacuole; promotes proteasome-dependent catabolite degradation of FBPase CAGL0K04301g 3,47 1,20E-05 FMP48 Putative protein of unknown function; the authentic, non-tagged protein is detected in highly purified mitochondria in high-throughput studies; induced by treatment with 8-methoxypsoralen and UVA irradiation CAGL0B00616g 3,46 3,39E-05 SPS22 Protein of unknown function, redundant with Sps2p for the organization of the beta-glucan layer of the spore wall CAGL0E06358g CgGPM1 3,46 1,18E-06 GPM1 Tetrameric phosphoglycerate mutase, mediates the conversion of 3-phosphoglycerate to 2-phosphoglycerate during glycolysis and the reverse reaction during gluconeogenesis CAGL0K03421g 3,39 2,14E-05 PGM2 Phosphoglucomutase, catalyzes the conversion from glucose-1-phosphate to glucose-6-phosphate, which is a key step in hexose metabolism; functions as the acceptor for a Glc-phosphotransferase CAGL0L08008g 3,39 1,40E-04 PMP1 Small single-membrane span proteolipid that functions as a regulatory subunit of the plasma membrane H(+)-ATPase Pma1p, forms unique helix and positively charged cytoplasmic domain that is able to specifically segregate phosphatidylserines CAGL0H02101g 3,31 8,50E-05 RTC3 Protein of unknown function involved in RNA metabolism; has structural similarity to SBDS, the human protein mutated in Shwachman-Diamond Syndrome (the yeast SBDS ortholog = SDO1); null mutation suppresses cdc13-1 temperature sensitivity Protein of unknown function; inhibits haploid invasive growth when overexpressed; synthetically lethal with phospholipase C (PLC1); expression induced by mild heatstress on a non-fermentable carbon source, upon entry into stationary phase and upon nitrogen deprivation; repressed by inosine and choline in an Opi1pdependent manner; highly conserved from bacteria to human; Memo, the human homolog, is an ErbB2 interacting protein with an essential function in cell motility Protein of unknown function; some similarity to AMP deaminases but lacks key catalytic residues and does not rescue purine nucleotide metabolic defect of quadruple aah1 ade8 amd1 his1 mutant; may regulate purine nucleotide homeostasis as overexpression in an AMD1 strain grown in adenine results in greatly reduced GDP and GTP intracellular levels; the authentic, non-tagged protein is detected in highly purified mitochondria in high-throughput studies; not an essential gene CAGL0G05357g 1,39 1,06E-03 YNL200C NADHX epimerase; catalyzes isomerization of (R)-and (S)-NADHX; homologous to AIBP in mammals and the N-terminal domain of YjeF in E.coli; enzyme is widespread in eukaryotes, prokaryotes and archaea; the authentic, non-tagged protein is detected in highly purified mitochondria in high-throughput studies Twinfilin, highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton, composed of two cofilin-like regions, localizes actin monomers to sites of rapid filament assembly CAGL0H09614g 1,23 5,89E-03 TIR1 Cell wall mannoprotein of the Srp1p/Tip1p family of serine-alanine-rich proteins; expression is downregulated at acidic pH and induced by cold shock and anaerobiosis; abundance is increased in cells cultured without shaking CAGL0K04257g 1,23 1,01E-03 RME1 Zinc finger protein involved in control of meiosis; prevents meiosis by repressing IME1 expression and promotes mitosis by activating CLN2 expression; directly repressed by a1-alpha2 regulator; mediates cell type control of sporulation CAGL0L09108g 1,22 1,53E-03 PDH1 Mitochondrial protein that participates in respiration, induced by diauxic shift; homologous to E. coli PrpD, may take part in the conversion of 2-methylcitrate to 2methylisocitrate CAGL0E06666g CgEPA2 1,22 2,14E-03 -Epithelial adhesion protein CAGL0H02695g 1,21 1,29E-03 GLG1 Self-glucosylating initiator of glycogen synthesis, also glucosylates n-dodecyl-beta-D-maltoside; similar to mammalian glycogenin CAGL0I03476g 1,21 8,12E-03 DHH1 Cytoplasmic DExD/H-box helicase, stimulates mRNA decapping; coordinates distinct steps in mRNA function and decay, interacts with both the decapping and deadenylase complexes, may have a role in mRNA export and translation; C-terminus of Dhh1p interacts with Ngr1p and promotes POR1, but not EDC1 mRNA decay 14-3-3 protein, major isoform; controls proteome at post-transcriptional level, binds proteins and DNA, involved in regulation of many processes including exocytosis, vesicle transport, Ras/MAPK signaling, and rapamycin-sensitive signaling CAGL0K04675g 1,19 1,74E-03 -Unknown CAGL0E01419g CgYPS2 1,18 1,81E-03 MKC7 GPI-anchored aspartyl protease, member of the yapsin family of proteases involved in cell wall growth and maintenance; shares functions with Yap3p and Kex2p CAGL0L10142g CgRSB1 1,18 2,64E-03 RSB1 Suppressor of sphingoid long chain base (LCB) sensitivity of an LCB-lyase mutation; putative integral membrane transporter or flippase that may transport LCBs from the cytoplasmic side toward the extracytoplasmic side of the membrane CAGL0H01287g 1,18 2,68E-03 SSD1 Translational repressor with a role in polar growth and wall integrity; regulated by Cbk1p phosphorylation to effect bud-specific translational control and localization of specific mRNAs; interacts with TOR pathway components; contains a functional N-terminal nuclear localization sequence and nucleocytoplasmic shuttling appears to be critical to Ssd1p function

CAGL0M08800g
CgYAP6 1,17 4,24E-03 YAP6 Basic leucine zipper (bZIP) transcription factor; physically interacts with the Tup1-Cyc8 complex and recruits Tup1p to its targets; overexpression increases sodium and lithium tolerance; computational analysis suggests a role in regulation of expression of genes involved in carbohydrate metabolism CAGL0M06457g 1,17 2,49E-03 GDT1 Putative protein of unknown function; expression is reduced in a gcr1 null mutant; GFP-fusion protein localizes to the vacuole; expression pattern and physical interactions suggest a possible role in ribosome biogenesis CAGL0J01892g CgPAN1 1,17 3,39E-03 PAN1 Part of actin cytoskeleton-regulatory complex Pan1p-Sla1p-End3p, associates with actin patches on the cell cortex; promotes protein-protein interactions essential for endocytosis; binds to and activates the Arp2/3 complex in vitro; previously thought to be a subunit of poly(A) ribonuclease CAGL0L06094g CgSTR3 1,17 6,84E-03 STR3 Peroxisomal cystathionine beta-lyase, converts cystathionine into homocysteine; may be redox regulated by Gto1p CAGL0L09691g 1,17 1,59E-03 PUT3 Transcriptional activator of proline utilization genes, constitutively binds PUT1 and PUT2 promoter sequences as a dimer and undergoes a conformational change to form the active state; differentially phosphorylated in the presence of different nitrogen sources; has a Zn(2)-Cys (6)  Ribosomal protein 51 (rp51) of the small (40s) subunit; nearly identical to Rps17Ap and has similarity to rat S17 ribosomal protein CAGL0M13563g -1,26 5,53E-03 GOT1 Homodimeric protein that is packaged into COPII vesicles and cycles between the ER and Golgi; involved in secretory transport but not directly required for aspects of transport assayed in vitro; may influence membrane composition CAGL0A01562g -1,26 8,39E-03 RPL24A Ribosomal protein L30 of the large (60S) ribosomal subunit, nearly identical to Rpl24Bp and has similarity to rat L24 ribosomal protein; not essential for translation but may be required for normal translation rate CAGL0K07436g -1,27 3,74E-03 YHM2 Carrier protein that exports citrate from and imports oxoglutarate into the mitochondrion, causing net export of NADPH reducing equivalents; also associates with mt nucleoids and has a role in replication and segregation of the mt genome CAGL0C04983g CgADO1 -1,28 6,84E-03 ADO1 Adenosine kinase, required for the utilization of S-adenosylmethionine (AdoMet); may be involved in recycling adenosine produced through the methyl cycle CAGL0H05511g - Cytosine deaminase, zinc metalloenzyme that catalyzes the hydrolytic deamination of cytosine to uracil; of biomedical interest because it also catalyzes the deamination of 5-fluorocytosine (5FC) to form anticancer drug 5-fluorouracil (5FU)

CAGL0A00627g
-1,53 7,63E-04 ERP6 Protein with similarity to Emp24p and Erv25p, member of the p24 family involved in ER to Golgi transport; the authentic, non-tagged protein is detected in highly purified mitochondria in high-throughput studies CAGL0B01122g CgSAM1 -1,54 1,02E-03 SAM1 S-adenosylmethionine synthetase, catalyzes transfer of the adenosyl group of ATP to the sulfur atom of methionine; one of two differentially regulated isozymes (Sam1p and Sam2p)

CAGL0D01716g
-1,63 1,36E-03 POL30 Proliferating cell nuclear antigen (PCNA), functions as the sliding clamp for DNA polymerase delta; may function as a docking site for other proteins required for mitotic and meiotic chromosomal DNA replication and for DNA repair