The Candida albicans Cdk8-dependent phosphoproteome reveals repression of hyphal growth through a Flo8-dependent pathway

Ssn3, also known as Cdk8, is a member of the four protein Cdk8 submodule within the multi-subunit Mediator complex involved in the co-regulation of transcription. In Candida albicans, the loss of Ssn3 kinase activity affects multiple phenotypes including cellular morphology, metabolism, nutrient acquisition, immune cell interactions, and drug resistance. In these studies, we generated a strain in which Ssn3 was replaced with a functional variant of Ssn3 that can be rapidly and selectively inhibited by the ATP analog 3-MB-PP1. Consistent with ssn3 null mutant and kinase dead phenotypes, inhibition of Ssn3 kinase activity promoted hypha formation. Furthermore, the increased expression of hypha-specific genes was the strongest transcriptional signal upon inhibition of Ssn3 in transcriptomics analyses. Rapid inactivation of Ssn3 was used for phosphoproteomic studies performed to identify Ssn3 kinase substrates associated with filamentation potential. Both previously validated and novel Ssn3 targets were identified. Protein phosphorylation sites that were reduced specifically upon Ssn3 inhibition included two sites in Flo8 which is a transcription factor known to positively regulate C. albicans morphology. Mutation of the two Flo8 phosphosites (threonine 589 and serine 620) was sufficient to increase Flo8-HA levels and Flo8 dependent transcriptional and morphological changes, suggesting that Ssn3 kinase activity negatively regulates Flo8.Under embedded conditions, when ssn3Δ/Δ and efg1Δ/Δ mutants were hyperfilamentous, FLO8 was essential for hypha formation. Previous work has also shown that loss of Ssn3 activity leads to increased alkalinization of medium with amino acids. Here, we show that the ssn3Δ/Δ medium alkalinization phenotype, which is dependent on STP2, a transcription factor involved in amino acid utilization, also requires FLO8 and EFG1. Together, these data show that Ssn3 activity can modulate Flo8 and its direct and indirect interactions in different ways, and underscores the potential importance of considering Ssn3 function in the control of transcription factor activities.

Bacterial origin of thymidylate and folate metabolism in Asgard Archaea

Little is known about the evolution and biosynthetic function of DNA precursor and the folate metabolism in the Asgard group of archaea. As Asgard occupy a key position in the archaeal and eukaryotic phylogenetic trees, we have exploited very recently emerged genome and metagenome sequence information to investigate these central metabolic pathways. Our genome-wide analyses revealed that the recently cultured Asgard archaeon Candidatus Prometheoarchaeum syntrophicum strain MK-D1 (Psyn) contains a complete folate-dependent network for the biosynthesis of DNA/RNA precursors, amino acids and syntrophic amino acid utilization. Altogether our experimental and computational data suggest that phylogenetic incongruences of functional folate-dependent enzymes from Asgard archaea reflect their persistent horizontal transmission from various bacterial groups, which has rewired the key metabolic reactions in an important and recently identified archaeal phylogenetic group. We also experimentally validated the functionality of the lateral gene transfer of Psyn thymidylate synthase ThyX. This enzyme uses bacterial-like folates efficiently and is inhibited by mycobacterial ThyX inhibitors. Our data raise the possibility that the thymidylate metabolism, required for de novo DNA synthesis, originated in bacteria and has been independently transferred to archaea and eukaryotes. In conclusion, our study has revealed that recent prevalent lateral gene transfer has markedly shaped the evolution of Asgard archaea by allowing them to adapt to specific ecological niches.

Fish Protein and Its Derivatives: Functionality, Biotechnology and Health Effects

Animal derived proteins provide the essential amino acid requirements of a healthy diet. Among animal proteins, fish proteins have a well-balanced amino acid composition that are imperative for beneficial synthesizing and utilizing proteins in the body. Lack or low levels of any amino acid reduce the other amino acid utilization proportionately. This is why plant-derived proteins are potentially at a disadvantage for use in the body since they have relatively low levels of the essential amino acids methionine and lysine. Health studies have revealed that fish protein is suggested as a weight-loss diet to obese patients.

Fish Protein and Its Derivatives: Functionality, Biotechnology and Health Effects

Animal derived proteins provide the essential amino acid requirements of a healthy diet. Among animal proteins, fish proteins have a well-balanced amino acid composition that are imperative for beneficial synthesizing and utilizing proteins in the body. Lack or low levels of any amino acid reduce the other amino acid utilization proportionately. This is why plant-derived proteins are potentially at a disadvantage for use in the body since they have relatively low levels of the essential amino acids methionine and lysine. Health studies have revealed that fish protein is suggested as a weight-loss diet to obese patients.

Metabolic and molecular signatures of improved growth in Atlantic salmon (Salmo salar) fed surplus levels of methionine, folic acid, vitamin B6 and B12 throughout smoltification

A moderate surplus of the 1C nutrients methionine, folic acid, vitamin B6 and B12 above dietary recommendations for Atlantic salmon has shown to improve growth and reduce hepatosomatic index in the on-growing salt water period when fed throughout smoltification. Metabolic properties and molecular mechanisms determining the improved growth are unexplored. Here, we investigate metabolic and transcriptional signatures in skeletal muscle taken before and after smoltification to acquire deeper insight into pathways and possible nutrient-gene-interactions. A control feed (Ctrl) or 1C nutrient surplus feed (1C+) were fed to Atlantic salmon six weeks prior to smoltification until three months after salt water transfer. Both metabolic and gene expression signatures revealed significant 1C nutrient-dependent changes already at pre-smolt, but differences intensified when analysing post-smolt muscle. Transcriptional differences revealed a lower expression of genes related to translation, growth, and amino acid metabolization in post-smolt muscle when fed additional 1C nutrients. The 1C+ group showed less free amino acid and putrescine levels, and higher methionine and glutathione (GSH) amounts in muscle. For Ctrl muscle, the overall metabolic profile suggests a lower amino acid utilization for protein synthesis, and increased methionine metabolization in polyamine and redox homeostasis, whereas transcription changes are indicative of compensatory growth regulation at local tissue level. These findings point to fine-tuned nutrient-gene-interactions fundamental for improved growth capacity through better amino acid utilization for protein accretion when salmon was fed additional 1C nutrients throughout smoltification. It also highlights potential nutritional programming strategies on improved post-smolt growth through 1C+ supplementation before and throughout smoltification.

The Candida albicans Cdk8-dependent phosphoproteome reveals repression of hyphal growth through a Flo8-dependent pathway

Ssn3, also known as Cdk8, is a member of the four protein Cdk8 submodule within the multi-subunit Mediator complex involved in the co-regulation of transcription. In Candida albicans , the loss of Ssn3 kinase activity affects multiple phenotypes including cellular morphology, metabolism, nutrient acquisition, immune cell interactions, and drug resistance. In these studies, we generated a strain in which Ssn3 was replaced with a functional variant of Ssn3 that can be rapidly and selectively inhibited by the ATP analog 3-MB-PP1 . Consistent with ssn3 null mutant and kinase dead phenotypes, inhibition of Ssn3 kinase activity promoted hypha formation. Furthermore, the increased expression of hypha-specific genes was the strongest transcriptional signal upon inhibition of Ssn3 in transcriptomics analyses. Rapid inactivation of Ssn3 was used for phosphoproteomic studies performed to identify Ssn3 kinase substrates associated with filamentation potential. Both previously validated and novel Ssn3 targets were identified. Protein phosphorylation sites that were reduced specifically upon Ssn3 inhibition included two sites in Flo8 which is a transcription factor known to positively regulate C. albicans morphology. Mutation of the two Flo8 phosphosites (threonine 589 and serine 620) was sufficient to increase Flo8-HA levels and Flo8 dependent activity, suggesting that Ssn3 kinase activity negatively regulates Flo8. Previous work has also shown that loss of Ssn3 activity leads to increased alkalinization of medium with amino acids. Here, we show that FLO8 and STP2 , a transcription factor involved in amino acid utilization, are required for ssn3 ?/? phenotype, but that loss of the Ssn3 phosphosites identified in Flo8 was not sufficient to phenocopy the ssn3 ?/? mutant. These data highlight the spectrum of processes affected by the modulation of Ssn3 activity and underscore the importance of considering Ssn3 function in the control of transcription factor activities.

Metabolic Regulation of Immune Responses to Mycobacterium tuberculosis: A Spotlight on L-Arginine and L-Tryptophan Metabolism

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a leading cause of death worldwide. Despite decades of research, there is still much to be uncovered regarding the immune response to Mtb infection. Here, we summarize the current knowledge on anti-Mtb immunity, with a spotlight on immune cell amino acid metabolism. Specifically, we discuss L-arginine and L-tryptophan, focusing on their requirements, regulatory roles, and potential use as adjunctive therapy in TB patients. By continuing to uncover the immune cell contribution during Mtb infection and how amino acid utilization regulates their functions, it is anticipated that novel host-directed therapies may be developed and/or refined, helping to eradicate TB.