La respuesta al estrés oxidante de Candida glabrata a través de la regulación génica de CTA1

Abstract

""Candida glabrata is an opportunistic pathogenic haploid yeast that is found as a commensal in healthy individuals but can cause severe infections in immunocompromised patients. C. glabrata has enzymatic and non-enzymatic mechanisms to counteract oxidative stress. C. glabrata has only one catalase (Cta1) which confers resistance to high concentrations of H2O2 in vitro. This suggests that the regulation of CTA1 expression might be important in understanding the virulence of this yeast. We did transcriptional fusions of the intergenic region between OYE2 and CTA1 and GFP as a reporter gene to map the CTA1 promoter, to identify cis-acting regulatory elements and to determine which transcription factors are important for the induction of CTA1 in the presence of oxidative stress. We generated 5' to 3' deletions of the intergenic region between OYE2 and CTA1 and analyzed the activity of the CTA1 promoter by FACS (Fluorescence-Activated Cell Sorting) in different genetic backgrounds (wild type, yap1Δ, skn7Δ, yap1Δ skn7Δ, msn2Δ, msn4Δ, msn2Δ msn4Δ). The basal promoter is located between -1 kb and -0.75 kb from the ATG of CTA1 and we found two cis-acting regulatory elements located between -4 kb and -3.3 kb, between -1.34 kb and -1 kb. In addition, we determined that both transcription factors, Yap1 and Skn7, are required for the induction of the CTA1 promoter in the presence of H2O2, while Msn2 and Msn4 are dispensable. Additionally, we performed a heterologous complementation assay with pPScCTA1::CgCTA1::3'UTRScCTA1 on different genetic backgrounds of S. cerevisiae: wild type, cta1Δ, ctt1Δ, cta1Δ ctt1Δ). Both in stationary phase and in log phase of growth, the different genetic backgrounds with the plasmid pPScCTA1::CgCTA1::3'UTRScCTA1 remarkably increase their resistance to H2O2., due to C. glabrata catalase higher enzymatic activity."

"Candida glabrata is an opportunistic pathogenic haploid yeast that is found as a commensal in healthy individuals but can cause severe infections in immunocompromised patients. C. glabrata has enzymatic and non-enzymatic mechanisms to counteract oxidative stress. C. glabrata has only one catalase (Cta1) which confers resistance to high concentrations of H2O2 in vitro. This suggests that the regulation of CTA1 expression might be important in understanding the virulence of this yeast. We did transcriptional fusions of the intergenic region between OYE2 and CTA1 and GFP as a reporter gene to map the CTA1 promoter, to identify cis-acting regulatory elements and to determine which transcription factors are important for the induction of CTA1 in the presence of oxidative stress. We generated 5' to 3' deletions of the intergenic region between OYE2 and CTA1 and analyzed the activity of the CTA1 promoter by FACS (Fluorescence-Activated Cell Sorting) in different genetic backgrounds (wild type, yap1Δ, skn7Δ, yap1Δ skn7Δ, msn2Δ, msn4Δ, msn2Δ msn4Δ). The basal promoter is located between -1 kb and -0.75 kb from the ATG of CTA1 and we found two cis-acting regulatory elements located between -4 kb and -3.3 kb, between -1.34 kb and -1 kb. In addition, we determined that both transcription factors, Yap1 and Skn7, are required for the induction of the CTA1 promoter in the presence of H2O2, while Msn2 and Msn4 are dispensable. Additionally, we performed a heterologous complementation assay with pPScCTA1