Dataset Information

Trypanosomatid selenophosphate synthetase structure, function and interaction with selenocysteine lyase.

ABSTRACT: Eukaryotes from the Excavata superphylum have been used as models to study the evolution of cellular molecular processes. Strikingly, human parasites of the Trypanosomatidae family (T. brucei, T. cruzi and L. major) conserve the complex machinery responsible for selenocysteine biosynthesis and incorporation in selenoproteins (SELENOK/SelK, SELENOT/SelT and SELENOTryp/SelTryp), although these proteins do not seem to be essential for parasite viability under laboratory controlled conditions. Selenophosphate synthetase (SEPHS/SPS) plays an indispensable role in selenium metabolism, being responsible for catalyzing the formation of selenophosphate, the biological selenium donor for selenocysteine synthesis. We solved the crystal structure of the L. major selenophosphate synthetase and confirmed that its dimeric organization is functionally important throughout the domains of life. We also demonstrated its interaction with selenocysteine lyase (SCLY) and showed that it is not present in other stable assemblies involved in the selenocysteine pathway, namely the phosphoseryl-tRNASec kinase (PSTK)-Sec-tRNASec synthase (SEPSECS) complex and the tRNASec-specific elongation factor (eEFSec) complex. Endoplasmic reticulum stress with dithiothreitol (DTT) or tunicamycin upon selenophosphate synthetase ablation in procyclic T. brucei cells led to a growth defect. On the other hand, only DTT presented a negative effect in bloodstream T. brucei expressing selenophosphate synthetase-RNAi. Furthermore, selenoprotein T (SELENOT) was dispensable for both forms of the parasite. Together, our data suggest a role for the T. brucei selenophosphate synthetase in the regulation of the parasite's ER stress response.

SUBMITTER: da Silva MTA

PROVIDER: S-EPMC7595633 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Trypanosomatid selenophosphate synthetase structure, function and interaction with selenocysteine lyase.

da Silva Marco Túlio Alves MTA Silva Ivan Rosa E IRE Faim Lívia Maria LM Bellini Natália Karla NK Pereira Murilo Leão ML Lima Ana Laura AL de Jesus Teresa Cristina Leandro TCL Costa Fernanda Cristina FC Watanabe Tatiana Faria TF Pereira Humberto D'Muniz HD Valentini Sandro Roberto SR Zanelli Cleslei Fernando CF Borges Júlio Cesar JC Dias Marcio Vinicius Bertacine MVB da Cunha Júlia Pinheiro Chagas JPC Mittra Bidyottam B Andrews Norma W NW Thiemann Otavio Henrique OH

PLoS neglected tropical diseases 20201005 10

Eukaryotes from the Excavata superphylum have been used as models to study the evolution of cellular molecular processes. Strikingly, human parasites of the Trypanosomatidae family (T. brucei, T. cruzi and L. major) conserve the complex machinery responsible for selenocysteine biosynthesis and incorporation in selenoproteins (SELENOK/SelK, SELENOT/SelT and SELENOTryp/SelTryp), although these proteins do not seem to be essential for parasite viability under laboratory controlled conditions. Selen ...[more]

PMID: 33017394

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Project description:The endospore-forming pathogen Clostridioides difficile is the leading cause of antibiotic-associated diarrhea and is a significant burden on the community and health care. C. difficile, like all forms of life, incorporates selenium into proteins through a selenocysteine synthesis pathway. The known selenoproteins in C. difficile are involved in a metabolic process that uses amino acids as the sole carbon and nitrogen source (Stickland metabolism). The Stickland metabolic pathway requires the use of two selenium-containing reductases. In this study, we built upon our initial characterization of the CRISPR-Cas9-generated selD mutant by creating a CRISPR-Cas9-mediated restoration of the selD gene at the native locus. Here, we use these CRISPR-generated strains to analyze the importance of selenium-containing proteins on C. difficile physiology. SelD is the first enzyme in the pathway for selenoprotein synthesis, and we found that multiple aspects of C. difficile physiology were affected (e.g., growth, sporulation, and outgrowth of a vegetative cell post-spore germination). Using transcriptome sequencing (RNA-seq), we identified multiple candidate genes which likely aid the cell in overcoming the global loss of selenoproteins to grow in medium which is favorable for using Stickland metabolism. Our results suggest that the absence of selenophosphate (i.e., selenoprotein synthesis) leads to alterations to C. difficile physiology so that NAD+ can be regenerated by other pathways. IMPORTANCE C. difficile is a Gram-positive, anaerobic gut pathogen which infects thousands of individuals each year. In order to stop the C. difficile life cycle, other nonantibiotic treatment options are in urgent need of development. Toward this goal, we find that a metabolic process used by only a small fraction of the microbiota is important for C. difficile physiology: Stickland metabolism. Here, we use our CRISPR-Cas9 system to "knock in" a copy of the selD gene into the deletion strain to restore selD at its native locus. Our findings support the hypothesis that selenium-containing proteins are important for several aspects of C. difficile physiology, from vegetative growth to spore formation and outgrowth postgermination.

Dataset Information

Trypanosomatid selenophosphate synthetase structure, function and interaction with selenocysteine lyase.

Publications

Trypanosomatid selenophosphate synthetase structure, function and interaction with selenocysteine lyase.

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