The selenophosphate synthetase, selD, is important for Clostridioides difficile physiology

The endospore-forming pathogen, Clostridioides difficile, is the leading cause of antibiotic-associated diarrhea and is a significant burden on the community and healthcare. 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 RNAseq, 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 lifecycle, other non-antibiotic treatment options are in urgent need of development. Towards 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 post-germination.

The selenophosphate synthetase, selD, is important for Clostridioides difficile physiology

The endospore-forming pathogen, Clostridioides difficile, is the leading cause of antibiotic-associated diarrhea and is a significant burden on the community and healthcare. 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 RNAseq, 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.

Selenocysteine in Trypanosoma evansi: Identification of the Genes selb, selc, seld, pstk, seltryp and the Selenophosphate Synthetase Protein

Selenoproteins have been described in all three domains of life and their function has been mainly associated with oxidative stress defense. Canonical elements required for selenoprotein production have been identified in members of the kinetoplastid group supporting the existence of a complete selenocysteine synthesis pathway in these organisms. Currently, nothing is known regarding the selenocysteine pathway in Trypanosoma evansi. In this study, we identified the expression of the elements selB, selC, selD, PSTK and selTRYP at the mRNA level in T. evansi. All translated proteins (selD, PSTK, selTRYP and selB) have the domains predicted and higher identity with Trypanosoma brucei. gambiense. The selenophosphate synthetase protein was localized in the cytoplasm. Our results support the existence of an active selenocysteine pathway in T. evansi.

Trypanosomatid selenophosphate synthetase structure, function and interaction with selenocysteine lyase

Early branching eukaryotes have been used as models to study the evolution of cellular molecular processes. Strikingly, human parasite 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 complexes involved in the selenocysteine pathway, namely the phosphoseryl-tRNASec kinase (PSTK)-Sec-tRNASec synthase (SEPSECS) and the tRNASec-specific elongation factor (eEFSec)-ribosome. Endoplasmic reticulum stress with ditiothreitol (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. Although selenoprotein T (SELENOT) was dispensable for both forms of the parasite, SELENOT-RNAi procyclic T. brucei cells were sensitive to DTT. Together, our data suggest a role for the T. brucei selenophosphate synthetase in regulation of the parasite’s ER stress response.SynopsisSelenium is both a toxic compound and a micronutrient. As a micronutrient, it participates in the synthesis of specific proteins, selenoproteins, as the amino acid selenocysteine. The synthesis of selenocysteine is present in organisms ranging from bacteria to humans. The protozoa parasites of the Trypanosomatidae family, that cause major tropical diseases, conserve the complex machinery responsible for selenocysteine biosynthesis and incorporation in selenoproteins. However, this pathway has been considered dispensable for the protozoa cells. This has intrigued us, and lead to question that if maintained in the cell it should be under selective pressure and therefore be necessary. Also, since the intermediate products of selenocysteine synthesis are toxic to the cell, it has been proposed that these compounds need to be sequestered from the cytoplasm. Therefore, extensive and dynamic protein-protein interactions must happen to deliver those intermediates along the pathway. In this study we have investigated the molecular and structural interactions of different proteins involved in selenocystein synthesis and describe its involvement in the endoplasmic reticulum protection to oxidative stress. Our results also show how the interaction of different proteins leads to the protection of the cell against the toxic effects of seleium compounds during selenocysteine synthesis.