Article Figures & Data
Figures
- FIG. 1.
C. glabrata, C. albicans, and S. cerevisiae LP resistance to H2O2. Saturated cultures of C. glabrata (C.g.) strain BG14 and CI MC7 and MC22 (A), C. albicans (C.a.) strain CAI4 and CI CA5 and CA7 (B), and S. cerevisiae (S.c.) strain W303 and CI YJM128 and YJM336 (C) were diluted with fresh medium (YPD) so that all strains reached an OD600 of 0.5 after seven doublings at 30°C. C. glabrata and C. albicans strains were divided and exposed to 0, 10, 20, 30, 40, 50, and 100 mM H2O2 and S. cerevisiae strains were exposed to 2, 4, 6, and 8 mM H2O2 for 3 h. For adaptation experiments, C. glabrata and C. albicans cells were pretreated for 1 h with 10 mM H2O2 and then with 100 mM H2O2 for 2 additional hours. After the treatment, H2O2 was removed by centrifugation. The cultures were resuspended in distilled water, and the OD600s were adjusted when needed to 0.5. Cultures were serially diluted, and each dilution was spotted onto YPD plates, ensuring that the same amounts of cells were plated. Plates were incubated at 30°C. See Materials and Methods.
- FIG. 2.
C. glabrata, C. albicans, and S. cerevisiae SP resistance to H2O2. Saturated cultures of C. glabrata (C.g.) strain BG14 and CI MC7 and MC22 (A), C. albicans (C.a.) strain CAI4 and CI CA5 and CA7 (B), and S. cerevisiae (S.c.) strain W303 and CI YJM128 and YJM336 (C) were diluted to an OD600 of 0.5 with spent medium from the same cultures. The cells were divided into aliquots and treated for 3 h with H2O2 at different concentrations: for C. glabrata, 500, 800, 1,000, and 1,500 mM; for C. albicans, 0, 50, 100, 300, and 500 mM; and for S. cerevisiae, 0, 10, 50, 100, and 200 mM. After the treatment, the cultures remained at an OD600 of 0.5, oxidant was removed by centrifugation, cells were resuspended in distilled water, and suspensions were diluted and spotted onto YPD plates. Plates were incubated at 30°C. See Materials and Methods.
- FIG. 3.
Regulation of the OSR to H2O2 in LP. The wt (BG14) and single, double, and triple mutants with yap1Δ, skn7Δ, msn2Δ, and msn4Δ mutations were grown and treated with H2O2 as described in the legend to Fig. 1. See Materials and Methods.
- FIG. 4.
Regulation of the OSR to H2O2 in SP. The wt (BG14) and single, double, and triple mutants with yap1Δ, skn7Δ, msn2Δ, and msn4Δ mutations were grown and treated with H2O2 as described in the legend to Fig. 2. See Materials and Methods.
- FIG. 5.
ScCta1p and CgCta1p alignment. ScCta1p and CgCta1p are 85% similar across the entire lengths of the proteins. Identical residues are boxed and shaded. SKF (indicated by boxes and asterisks) is the peroxisomal targeting signal.
- FIG. 6.
Analysis of Cta1p in the OSR. Experiments with both LP and SP wt (BG14), cta1Δ, yap1Δ skn7Δ msn4Δ, and yap1Δ skn7Δ msn2Δ msn4Δ cells were performed as described in the legends to Fig. 1 and 2. See Materials and Methods.
- FIG. 7.
Growth curves of the cta1Δ mutant versus the wt (BG14) at 37°C.
- FIG. 8.
Cta1p is dispensable in vivo. Numbers of CFU in kidney, spleen, and liver tissues 7 days postinfection with the wt (BG462) and the cta1Δ mutant (CGM351) are shown. Individual datum points represent results for individual mice in groups of 10 mice. The bars indicate the geometric mean for each group. See Materials and Methods.
Tables
- TABLE 1.
Strains used in this study
Strain Parent Genotype and/or description Reference or source E. coli strains DH10B F−mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 deoR recA1 endA1 araD139 Δ(ara,leu)7697 galU galK λ−rpsL nupG 7 JM109 recA1 endA1 gyrA96 thi hsdR17(rK− mK+) relA1 supE44 Δ(lac-proAB) [F′ traD36 proAB lacIqZΔM15] 77 S. cerevisiae strains W303 MAT a ura3-1 leu2-3,112 his3-11,15 trp1-1 can1-100 ade2-1 ade3::hisG 51 YJM128 Clinical isolate 12 YJM336 Clinical isolate 12 C. albicans strains CAI4 ura3Δ::imm434/ura3Δ::imm434 25 CA5 Clinical isolate Lab collection CA7 Clinical isolate Lab collection C. glabrata strains MC7 Clinical isolate Lab collection MC22 Clinical isolate Lab collection BG2 Clinical isolate (strain B) 22 BG14 BG2 ura3Δ::Tn903 G418r; wt Ura− strain used in this study 15 BG462 BG14 URA3 18 BG1739 (msn2Δ mutant) BG14 ura3Δ::Tn903 G418rmsn2Δ Hygs Ura− pRD96/BcgI R. Domergue and B. Cormack BG1740 (msn4Δ mutant) BG14 ura3Δ::Tn903 G418rmsn4Δ Hygs Ura− pRD97/BcgI R. Domergue and B. Cormack BG1742 (msn2Δ msn4Δ mutant) BG1739 ura3Δ::Tn903 G418rmsn2Δ msn4Δ::hph Hygr R. Domergue and B. Cormack CGM295 (cta1Δ mutant) BG14 ura3Δ::Tn903 G418rcta1Δ::hph Hygr pCV15/BcgI This work CGM297 (yap1Δ mutant) BG14 ura3Δ::Tn903 G418ryap1Δ::hph Hygr pCV17/BcgI This work CGM306 (skn7Δ mutant) BG14 ura3Δ::Tn903 G418rskn7Δ::hph Hygr pCV21/BcgI This work CGM308 (yap1Δ mutant) CGM297 ura3Δ::Tn903 G418ryap1Δ Hygs Ura− This work CGM310 (yap1Δ skn7Δ mutant) CGM308 ura3Δ::Tn903 G418ryap1Δ skn7Δ::hph Hygr This work CGM351 (cta1Δ mutant) CGM295 URA3 cta1Δ::hph Hygr This work CGM385 (yap1Δ skn7Δ mutant) CGM310 ura3Δ::Tn903 G418ryap1Δ skn7Δ Hygs Ura− This work CGM386 (yap1Δ skn7Δ msn2Δ mutant) CGM385 ura3Δ::Tn903 G418ryap1Δ skn7Δ msn2Δ::hph Hygr This work CGM388 (yap1Δ skn7Δ msn4Δ mutant) CGM385 ura3Δ::Tn903 G418ryap1Δ skn7Δ msn4Δ::hph Hygr This work CGM480 (yap1Δ skn7Δ msn4Δ mutant) CGM388 ura3Δ::Tn903 G418ryap1Δ skn7Δ msn4Δ Hygs Ura− This work CGM537 (yap1Δ skn7Δ msn4Δ msn2Δ mutant) CGM480 ura3Δ::Tn903 G418ryap1Δ skn7Δ msn4Δ msn2Δ::hph Hygr This work - TABLE 2.
Plasmids used in this study
Plasmid Description and/or relevant genotype Reference or source Plasmids for cloning, construction, and marker removal pGEM-T Cloning vector; Kmr Apr Promega pAP599 Cloning vector with 2 FRT direct repeats flanking the hygromycin marker [FRT-PPGK::hph::(3′ UTR of HIS3)-FRT] for construction of multiple mutants; URA3 Hygr Ampr 19 pMZ17 Replicative vector expressing ScFLP1 (recombinase gene) for removing the hygromycin marker; PPGK::FLP1::(3′ UTR of HIS3) CgCEN ARS Ampr Cormack lab collection pBC34.1 pUC19::CgURA3; 2.2-kb PstI fragment; Apr 15 Plasmids for deletion pCV15 cta1Δ A 0.906-kb SpeI/BamHI PCR fragment (corresponding to primers 57 and 58) carrying the promoter region of CTA1 and a 0.682-kb HindIII/KpnI PCR fragment (corresponding to primers 60 and 61) carrying the 3′ UTR of CTA1 were cloned into pAP599 Apr This work pCV17 yap1Δ A 0.846-kb XhoI/HindIII PCR fragment (corresponding to primers 7 and 8) carrying the promoter region of YAP1 and a 0.652-kb BamHI/SacI PCR fragment (corresponding to primers 11 and 12) carrying the 3′ UTR of YAP1 were cloned into pAP599 Apr This work CV21 skn7Δ A 0.875-kb KpnI/XhoI PCR fragment (corresponding to primers 4 and 5) carrying the promoter region of SKN7 and a 0.929-kb BamHI/SacI PCR fragment (corresponding to primers 1 and 2) carrying the 3′ UTR of SKN7 were cloned into pAP599 Apr This work pRD96 msn2Δ A 0.691-kb KpnI/XhoI PCR fragment (corresponding to primers 2984 and 2985) carrying the promoter region of MSN2 and a 0.520-kb BamHI/SacI PCR fragment (corresponding to primers 2986 and 2987) carrying the 3′ UTR of MSN2 were cloned into pAP599 Apr R. Domergue and B. Cormack pRD97 msn4Δ A 0.564-kb KpnI/XhoI PCR fragment (corresponding to primers 2990 and 2991) carrying the promoter region of MSN4 and a 0.535-kb BamHI/SacI PCR fragment (corresponding to primers 2992 and 2993) carrying the 3′ UTR of MSN4 were cloned into pAP599 Apr R. Domergue and B. Cormack - TABLE 3.
Oligonucleotides used in this study
Primer Sequencea Description or restriction site(s) 13 GTTGTAAAACGACGGCCAGTG pUC forward 17 GGAAACAGCTATGACCATG pUC reverse 1 CGCGGATCCAAGTATACTGCTATGAGCTAC BamHI 2 CAAGGAGCTCTTGTGCAGCGTGTAAGATATGAATCAAGTGAT SacI/BsgI 4 TCAGATCTCGAGTTGACCGTGACCGAAAC XhoI 5 CCGGGTACCTTGTGCAGTCGAAGTTAGAGTGCCCTATTC KpnI/BsgI 7 AATCTCGAGTTGTGCAGTGCGGGTAACAATTCTCGGCG XhoI/BsgI 8 CCCAAGCTTTTACTTCCTAGTTCTTGTCTC HindIII 11 CGCGGATCCTGTCTATATTATCTCGGTAGATC BamHI 12 CAAGGAGCTCTTGTGCAGTCAACTCATAGATCACAACATTAACAC SacI/BsgI 57 CGCGGATCCTCAATTGTGGGAAGTTATCTAATAAGCC BamHI 58 CACTACTAGTTAAACACTTGTAGGAG SpeI 60 CCCAAGCTTTTGAACCACGTAAAGTGCTGT HindIII 61 CCGGGTACCTTGTGCAGCCTGTAAGGACTTCTAAACC KpnI/BsgI 2984 GGTACCCGAGGGAGTTGCCTTCACTATGTGCGT TGAG KpnI/BcgI 2985 CTCGAGCTGTTCTTGTTGATCTGTGTTTGGT XhoI 2986 GGATTCTGACAGTGTTCCTTATTTTATCTAG BamHI 2987 GAGCTCCGACCGCGGTGCATCTGTTACCAGGTTAGCC SacI/BcgI 2990 GGTACCCGACTCCTGTGCCTTGTCGTACCAGAGAAAC KpnI/BcgI 2991 CTCGAGGAACAGGAATGGACACTAATATATA XhoI 2992 GGATCCCCATTCTTTATTTTATTTTCTGCTA BamHI 2993 GAGCTCCGAGGCATCTGCTAATAATTTTCCGTTTCAA SacI/BcgI ↵ a Restriction sites are indicated in boldface.
