Multiple Evolutionarily Conserved Domains of Cap2 Are Required for Promoter Recruitment and Iron Homeostasis Gene Regulation

ABSTRACT Iron is required for growth and metabolism by virtually all organisms. The human fungal pathogen Candida albicans has evolved multiple strategies to acquire iron. The Cap2/Hap43 transcriptional regulator, essential for robust virulence of C. albicans, controls iron homeostasis gene expressi...

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Autores principales:	Manjit Kumar Srivastav, Neha Agarwal, Krishnamurthy Natarajan
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	Candida albicans Cap2/Hap43 iron homeostasis transcriptional regulation Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/81cfaa1044d546ef9c1b9aef4feb6e49
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spelling	oai:doaj.org-article:81cfaa1044d546ef9c1b9aef4feb6e492021-11-15T15:25:50ZMultiple Evolutionarily Conserved Domains of Cap2 Are Required for Promoter Recruitment and Iron Homeostasis Gene Regulation10.1128/mSphere.00370-182379-5042https://doaj.org/article/81cfaa1044d546ef9c1b9aef4feb6e492018-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00370-18https://doaj.org/toc/2379-5042ABSTRACT Iron is required for growth and metabolism by virtually all organisms. The human fungal pathogen Candida albicans has evolved multiple strategies to acquire iron. The Cap2/Hap43 transcriptional regulator, essential for robust virulence of C. albicans, controls iron homeostasis gene expression by promoter binding and repression of iron utilization genes. The expression of iron uptake genes is also dependent on Cap2, although Cap2 was not recruited to its promoters. Cap2, bearing the conserved bipartite HAP4L-bZIP domain, also contains multiple blocks of amino acids that form the highly conserved carboxyl-terminal region. In this study, we sought to identify the requirements of the different domains for Cap2 function. We constructed a series of mutants bearing either point mutations or deletions in the conserved domains and examined Cap2 activity. Deletion of the highly conserved extreme C-terminal region did not impair expression of Cap2 mutant protein but impaired cell growth and expression of iron homeostasis genes under iron-depleted conditions. Mutations in the amino-terminal HAP4L and basic leucine zipper (bZIP) domains also impaired growth and gene expression. Furthermore, chromatin immunoprecipitation (ChIP) assays showed that the HAP4L domain and the bZIP domain are both essential for Cap2 recruitment to ACO1 and CYC1 promoters. Unexpectedly, the C-terminal conserved region was also essential for Cap2 promoter recruitment. Thus, our results suggest that Cap2 employs multiple evolutionarily conserved domains, including the C-terminal domain for its transcriptional activity. IMPORTANCE Iron is an essential micronutrient for living cells. Candida albicans, the predominant human fungal pathogen, thrives under diverse environments with vastly different iron levels in the mammalian host. Therefore, to tightly control iron homeostasis, C. albicans has evolved a set of transcriptional regulators that cooperate to either upregulate or downregulate transcription of iron uptake genes or iron utilization genes. Cap2/Hap43, a critical transcriptional regulator, contains multiple conserved protein domains. In this study, we carried out mutational analyses to identify the functional roles of the conserved protein domains in Cap2. Our results show that the bZIP, HAP4L, and the C-terminal domain are each required for Cap2 transcriptional activity. Thus, Cap2 employs multiple, disparate protein domains for regulation of iron homeostasis in C. albicans.Manjit Kumar SrivastavNeha AgarwalKrishnamurthy NatarajanAmerican Society for MicrobiologyarticleCandida albicansCap2/Hap43iron homeostasistranscriptional regulationMicrobiologyQR1-502ENmSphere, Vol 3, Iss 4 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Candida albicans Cap2/Hap43 iron homeostasis transcriptional regulation Microbiology QR1-502
spellingShingle	Candida albicans Cap2/Hap43 iron homeostasis transcriptional regulation Microbiology QR1-502 Manjit Kumar Srivastav Neha Agarwal Krishnamurthy Natarajan Multiple Evolutionarily Conserved Domains of Cap2 Are Required for Promoter Recruitment and Iron Homeostasis Gene Regulation
description	ABSTRACT Iron is required for growth and metabolism by virtually all organisms. The human fungal pathogen Candida albicans has evolved multiple strategies to acquire iron. The Cap2/Hap43 transcriptional regulator, essential for robust virulence of C. albicans, controls iron homeostasis gene expression by promoter binding and repression of iron utilization genes. The expression of iron uptake genes is also dependent on Cap2, although Cap2 was not recruited to its promoters. Cap2, bearing the conserved bipartite HAP4L-bZIP domain, also contains multiple blocks of amino acids that form the highly conserved carboxyl-terminal region. In this study, we sought to identify the requirements of the different domains for Cap2 function. We constructed a series of mutants bearing either point mutations or deletions in the conserved domains and examined Cap2 activity. Deletion of the highly conserved extreme C-terminal region did not impair expression of Cap2 mutant protein but impaired cell growth and expression of iron homeostasis genes under iron-depleted conditions. Mutations in the amino-terminal HAP4L and basic leucine zipper (bZIP) domains also impaired growth and gene expression. Furthermore, chromatin immunoprecipitation (ChIP) assays showed that the HAP4L domain and the bZIP domain are both essential for Cap2 recruitment to ACO1 and CYC1 promoters. Unexpectedly, the C-terminal conserved region was also essential for Cap2 promoter recruitment. Thus, our results suggest that Cap2 employs multiple evolutionarily conserved domains, including the C-terminal domain for its transcriptional activity. IMPORTANCE Iron is an essential micronutrient for living cells. Candida albicans, the predominant human fungal pathogen, thrives under diverse environments with vastly different iron levels in the mammalian host. Therefore, to tightly control iron homeostasis, C. albicans has evolved a set of transcriptional regulators that cooperate to either upregulate or downregulate transcription of iron uptake genes or iron utilization genes. Cap2/Hap43, a critical transcriptional regulator, contains multiple conserved protein domains. In this study, we carried out mutational analyses to identify the functional roles of the conserved protein domains in Cap2. Our results show that the bZIP, HAP4L, and the C-terminal domain are each required for Cap2 transcriptional activity. Thus, Cap2 employs multiple, disparate protein domains for regulation of iron homeostasis in C. albicans.
format	article
author	Manjit Kumar Srivastav Neha Agarwal Krishnamurthy Natarajan
author_facet	Manjit Kumar Srivastav Neha Agarwal Krishnamurthy Natarajan
author_sort	Manjit Kumar Srivastav
title	Multiple Evolutionarily Conserved Domains of Cap2 Are Required for Promoter Recruitment and Iron Homeostasis Gene Regulation
title_short	Multiple Evolutionarily Conserved Domains of Cap2 Are Required for Promoter Recruitment and Iron Homeostasis Gene Regulation
title_full	Multiple Evolutionarily Conserved Domains of Cap2 Are Required for Promoter Recruitment and Iron Homeostasis Gene Regulation
title_fullStr	Multiple Evolutionarily Conserved Domains of Cap2 Are Required for Promoter Recruitment and Iron Homeostasis Gene Regulation
title_full_unstemmed	Multiple Evolutionarily Conserved Domains of Cap2 Are Required for Promoter Recruitment and Iron Homeostasis Gene Regulation
title_sort	multiple evolutionarily conserved domains of cap2 are required for promoter recruitment and iron homeostasis gene regulation
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/81cfaa1044d546ef9c1b9aef4feb6e49
work_keys_str_mv	AT manjitkumarsrivastav multipleevolutionarilyconserveddomainsofcap2arerequiredforpromoterrecruitmentandironhomeostasisgeneregulation AT nehaagarwal multipleevolutionarilyconserveddomainsofcap2arerequiredforpromoterrecruitmentandironhomeostasisgeneregulation AT krishnamurthynatarajan multipleevolutionarilyconserveddomainsofcap2arerequiredforpromoterrecruitmentandironhomeostasisgeneregulation
_version_	1718427959901028352

Multiple Evolutionarily Conserved Domains of Cap2 Are Required for Promoter Recruitment and Iron Homeostasis Gene Regulation

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