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The Yak1 kinase is involved in the initiation and maintenance of hyphal growth in Candida albicans

Sophie Goyard^*, Philipp Knechtle^*,†, Murielle Chauvel^*, Adeline Mallet^‡, Marie-Christine Prévost^‡, Caroline Proux^§, Jean-Yves Coppée^§, Patrick Schwartz^||, Françoise Dromer^||, Hyunsook Park^¶, Scott Filler^¶, Guilhem Janbon^|| and Christophe d'Enfert^*,#

^*Unité Biologie et Pathogénicité Fongiques, INRA USC2019, ^‡Plate-forme de Microscopie Ultrastructurale, ^§Plate-Forme 2 – Puces à ADN, Pasteur Génopole Ile-de-France, and ^||Unité de Mycologie Moléculaire, CNRS URA3012, Institut Pasteur, Paris, France
^¶Department of Medicine, Los Angeles Biomedical Research Institute, Harbor-UCLA Medical Center, Torrance, California, United States of America, and David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.

^#Corresponding author : Christophe d’Enfert
Unité Biologie et Pathogénicité Fongiques
Institut Pasteur
25, rue du Docteur Roux, 75724 Paris Cedex 15, France
Tel: +33 (0)1 40 61 32 57, Fax: +33 (0)1 45 68 89 38, E-mail: denfert@pasteur.fr

Members of the dual-specificity tyrosine-phosphorylated and regulated kinases (DYRK) family perform a variety of functions in eukaryotes. We employed gene disruption, targeted pharmacologic inhibition, and genome-wide transcriptional profiling to dissect the function of the Yak1 DYRK in the human fungal pathogen, Candida albicans. C. albicans strains with mutant yak1 alleles showed defects in the yeast-to-hypha transition and in maintaining hyphal growth. They also could not form biofilms. Despite their in vitro filamentation defect, C. albicans yak1Δ/yak1Δ mutants remained virulent in animal models of systemic and oropharyngeal candidiasis. Transcriptional profiling showed that Yak1 was necessary for the up-regulation of only a subset of hypha-induced genes. Although downstream targets of the Tec1 and Bcr1 transcription factors were down-regulated in the yak1Δ/yak1Δ mutant, TEC1 and BCR1 were not. Furthermore, 63% of Yak1-dependent, hypha specific genes have been reported to be negatively regulated by the transcriptional repressor, Tup1. These results suggest that Yak1 may function in the Tup1 pathway to govern hyphal emergence and maintenance.

Total RNA was prepared from 70 ml cultures of strain SC5314 (YAK1/YAK1) and CEC362 (yak1Δ/yak1Δ). Saturation culture grown in YPD were diluted to 2x10⁶ cells ml^-1 and grown for 1 h in YPD at 30°C (Yeast phase) or grown for 1h in Lee medium at 37°C (Hyphal phase). Total RNA was isolated using Qiagen RNAeasy according to the manufacturer's instructions. Total RNA samples (5 µg) were indirectly labelled using Atlas PowerScript Fluorescent Labelling kit (Clontech) with 1 µg oligo dT (Invitrogen), according to the conditions recommended by the manufacturer. cDNAs were coupled with cyanines using Cy3 Mono-Reactive Dye or Cy5 Mono-Reactive Dye (GE Healthcare). Fluorescent cDNAs were then purified with the Fluortrap matrix (Clontech). Equal quantities of Cy3 and Cy5-labelled cDNAs were mixed and concentrated by Microcon YM-30 (Millipore). Purified cDNAs were hybridized with microarrays containing approx. 6,000 C. albicans probes representing nearly the whole genome according to the conditions recommended by the manufacturer (Eurogentec, Seraing, Belgium). Arrays were scanned with an Axon 4000a scanner with fixed PMT (PMT = 550 for Cy3 and 650 for Cy5). Four comparisons were performed: Hyphal phase wild-type cells vs Yeast phase wild-type cells; Hyphal phase mutant cells vs Yeast phase mutant cells; Hyphal phase wild-type cells vs Hyphal phase mutant cells; Yeast phase wild-type cells vs Yeast phase mutant cells. For each comparison, two biological replicates were used and each biological replicate was subjected to technical replicates with dye-swaps. Data were acquired and analyzed by Genepix Pro 5.0 (Axon Instrument). Data normalization using Lowess and analysis were performed using GeneSpring (Silicon Genetics, Redwood City, CA). The p values for differentially expressed genes between the compared strains or conditions were adjusted with Benjamini-Hochberg’s transformation.
These data were provided by Sophie Goyard and Christophe d'Enfert, Unité Biologie et Pathogénicité Fongiques, Institut Pasteur, France.

 

Experimental data

Species	Candida albicans
Strains	wild type : SC5314, Gillum et al., 1984 yak1Δ: CEC362 (yak1::HIS1/yak1::ARG4, ura3::?imm434/ura3::?imm434, arg4::hisG/ arg4::hisG, his1::hisG/ his1::hisG, RPS10/RPS10::CIp10-URA3), this study
Growth conditions	Saturation culture grown in YPD were diluted to 2x106 cells ml-1 and grown for 1 h in YPD at 30°C (Yeast phase) or grown for 1h in Lee medium at 37°C (Hyphal phase).
RNA extraction	Cells were harvested by centrifufation (yeasts) or filtration (hyphae) and frozen in dry ice.  Total RNA was isolated using Qiagen RNAeasy according to the manufacturer's instructions.
Labelling	Indirect labelling with Atlas PowerScript Fluorescent Labelling kit (Clontech) and Cy3/Cy5 Mono-Reactive Dye (GE Healthcare) ; purification with fluortrap matrix (Clontech)
Microarray	Glass microarrays with probes for 5907 C. albicans ORFs as described.
Microarray manufacturer	Eurogentec SA
Hybridization method	As described in SOPs.
Imaging & Data capture	Genepix Pro 5.0 (Axon Instrument)
Normalization Method	Intensity dependent (Lowess) normalisation by GeneSpring 7.0 (Silicon Genetics)
Quality analysis	In GenePix, spots containing dust or dye smears that would give artificially high signals were flagged manually;  GeneSpring did not use these data in further analysis.  Genes that were significantly regulated were identified using GeneSpring 7.0 (Silicon Genetics). The p values for differentially expressed genes between the compared strains or conditions were adjusted with Benjamini-Hochberg’s transformation.

 

 

Microarray Data

Experiment	Biological replicate	Cy3 labeling	Cy5 labeling	Slide #	Batch	Name of .txt files
						download all .txt files
Wild-type Hypha vs Yeast	1	Wild-type Hypha	Wild-type Yeast	5	B010G	WTH550cy3_WTL650_0005.gpr
	1	Wild-type Hypha	Wild-type Yeast	30	B010G	WTH550cy3_WTL650_0030.gpr
	2	Wild-type Hypha	Wild-type Yeast	20	B010G	WTH550cy3_WTL650_0020.gpr
	1	Wild-type Yeast	Wild-type Hypha	3	B010G	WTL550cy3_WTH650_0003.gpr
	1	Wild-type Yeast	Wild-type Hypha	24	B010G	WTL550cy3_WTH650_0024.gpr
	2	Wild-type Yeast	Wild-type Hypha	18	B010G	WTL550cy3_WTH650_0018.gpr
Hypha Wild-type vs yak1Δ	1	Wild-type Hypha	yak1Δ Hypha	28	B010G	WTH550cy3_YakH650_0028.gpr
	2	Wild-type Hypha	yak1Δ Hypha	12	B010G	WTH550cy3_YakH650_0012.gpr
	2	Wild-type Hypha	yak1Δ Hypha	15	B010G	WTH550cy3_YakH650_0015.gpr
	1	yak1Δ Hypha	Wild-type Hypha	1	B010G	YakH550cy3_WTH650_0001.gpr
	1	yak1Δ Hypha	Wild-type Hypha	23	B010G	YakH550cy3_WTH650_0023.gpr
	2	yak1Δ Hypha	Wild-type Hypha	11	B010G	YakH550cy3_WTH650_0011.gpr
	2	yak1Δ Hypha	Wild-type Hypha	14	B010G	YakH550cy3_WTH650_0014.gpr
yak1Δ Hypha vs Yeast	1	yak1Δ Hypha	yak1Δ Yeast	21	B010G	YakH550cy3_YakL650_0021.gpr
	1	yak1Δ Hypha	yak1Δ Yeast	26	B010G	YakH550cy3_YakL650_0026.gpr
	2	yak1Δ Hypha	yak1Δ Yeast	17	B010G	YakH550cy3_YakL650_0017.gpr
	1	yak1Δ Yeast	yak1Δ Hypha	29	B010G	YakL550cy3_YackH650_0029.gpr
	1	yak1Δ Yeast	yak1Δ Hypha	22	B010G	YakL550cy3_YakH650_0022.gpr
	2	yak1Δ Yeast	yak1Δ Hypha	16	B010G	YakL550cy3_YakH650_0016.gpr
Yeast Wild-type vs yak1Δ	1	Wild-type Yeast	yak1Δ Yeast	25	B010G	WTL550cy3_YakL650_0025.gpr
	2	Wild-type Yeast	yak1Δ Yeast	13	B010G	WTL550cy3_YakL650_0013.gpr
	1	yak1Δ Yeast	Wild-type Yeast	4	B010G	YakL550cy3_WTL650_0004.gpr
	1	yak1Δ Yeast	Wild-type Yeast	27	B010G	YakL550cy3_WTL650_0027.gpr
	2	yak1Δ Yeast	Wild-type Yeast	19	B010G	YakL550cy3_WTL650_0019.gpr

 

 

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