Synthesis Pathways Of Cysteine And Mechanism For Alleviating Heavy Metal Stress In Tetrahymena Thermophila

Cysteine,a sulfur-containing amino acid,is implicated in important biological processes,and plays a critical role in the structure,stability,and catalytic functions of many proteins.Cysteine is synthesized through two different pathways,the de novo pathway and the reverse transsulfuration pathway.The de novo pathway mainly exists in bacteria and plants,while in fungi and higher animals,cysteine biosynthesis proceeds through the reverse transsulfuration pathway.Interestingly,cysteine is produced through the de novo and reverse transsulfuration pathways in some parasitic protozoa,such as Leishmania major and Trypanosoma cruzi.Tetrahymena thermophila is a unicellular,free-living ciliate protozoan in fresh water,which has sensitive environmental response mechanisms and subtle metabolic pathway.However,the mechanism of cellular cysteine metabolism and responsive mechanisms to environmental stress are less clear.In this study,we identified genes involved in cysteine synthesis and systematically analyzed the biological functions of its metabolite H₂S,which revealed the cysteine synthesis pathways in free-living protists and the mechanism of tolerance to environmental stress.The main results are obtained as following:1.Two cysteine synthase gene homologues,Tt CSA1 and Tt CSA2,were identified based on the Tetrahymena Genome Database.Tt CSA1 showed high expression level at log-phase,and decreased at starvation and during the sexual developmental stage,while Tt CSA2 showed high expression levels at log-phase and sexual development stage,and low expression level at starvation stage.Tt Csa1 and Tt Csa2 contained conserved Lys48 and Lys77 for PLP covalent binding.Tt Csa1 was a cytoplasmic enzyme,while Tt Csa2 was localized around the mitochondrial outer membrane throughout different developmental stages.Tt Csa2 was also localized into degraded parental macronucleus and around new developing macronucleus and micronucleus at the anlagen stage.However,the truncated N-terminal signal peptide mutant Tt Csa2-?N23 lost the ability to target the outer membrane.Tt CSA1 and Tt CSA2 were synthesized according to Escherichia coli codon preference.His-Tt Csa1 and His-Tt Csa2 were expressed in E.coli.The optimal temperature and pH of purified Tt Csa1 and Tt Csa2 was 37?and8.5 using O-acetylserine and H₂S as substrates.They both showed O-acetylserine sulfhydrylase(OASS)and serine sulfhydrylase(SS)activities.Besides,Tt Csa1 also catalyzed H₂S producing.The knockout plasmids of Tt CSA1 and Tt CSA2 were constructed.The cysteine and GSH contents decreased in the csa1KO and csa2KD mutants.Furthermore,the cell proliferation analysis under the stress of Cd and Cu showed that the IC50 of csa1KO and csa2KD mutants significantly decreased.The results indicated that Tt Csa1 and Tt Csa2 participate in cysteine synthesis through de novo pathway,and increase heavy metal detoxicity in T.thermophila.2.A cystathionine?-synthase homologous gene Tt CBS1 and a cystathionine?-lyase homologous gene Tt CGL1 were identified based on the Tetrahymena Genome Database.Tt CBS1 showed high expression level at vegetative growth and decreased during starvation and sexual reproduction.The structural model revealed that Tt Cbs1 was composed of an N-terminal heme domain,a C-terminal Bateman module and a central catalytic core,which possessed the conserved PLP-binding pocket.Tt Cbs1was localized in cytoplasm.Tt CBS1 was synthesized according to E.coli codon preference,and His-Tt Cbs1 was expressed in E.coli.The optimal temperature and pH of purified Tt Cbs1 was 37?and 8.5 using serine and homocysteine as substrates to synthesize cystathionine.Tt Cbs1 showed CBS activity with the highest velocity and the k_cat values for serine and homocysteine were 2.97 and 2.74 s^-1,respectively.Tt Cbs1 possessed SS activity but not OASS activity in vitro,and also catalyzed the production of H₂S through?-replacement or?-elimination reaction.The cbs1KD mutant affected cysteine and GSH synthesis,indicating that Tt Cbs1 participates in reverse transsulfuration pathway to synthesize cysteine.The expression level of Tt CSA1 and Tt CSA2 was upregulated in cbs1KD mutant,indicating the two cysteine synthesis pathways in T.thermophila cells have functional compensatory effects.3.Cd has deleterious effects on different organisms.The exposure of 30?M Cd resulted in T.thermophila growth inhibition,and malondialdehyde(MDA)content considerably increase.Exogenous NaHS(donor of H₂S,70?M)inhibited Cd accumulation and promoted intracellular formation of Cd S nanoparticles in T.thermophila.H₂S significantly alleviated the Cd-induced toxicity by upregulating GSH content,SOD and CAT activities for detoxification of ROS.In addition,the molecular roles of exogenously H₂S application were explored by RNA sequencing under Cd stress in T.thermophila.Comparative transcriptome analysis showed that the expression levels of 9152 genes changed under Cd stress(4658 upregulated and 4494 downregulated).However,only 1359 genes were differentially expressed with NaHS treatment under Cd stress(1087 upregulated and 272downregulated).The functional categories of the differentially expressed genes(DEGs)by gene ontology(GO)revealed that the transcripts involved in the oxidation–reduction process,oxidoreductase activity,glutathione peroxidase activity,and cell redox homeostasis were the considerable enrichments between Cd stress and NaHS treatment under Cd stress.Kyoto Encyclopedia of Genes and Genomes(KEGG)indicated that the carbon metabolism,glutathione metabolism,metabolism of xenobiotics by cytochrome P450,and ABC transporters were significantly differentially expressed components between Cd stress and NaHS treatment under Cd stress in T.thermophila.Endogenous cysteine and H₂S levels showed a dose-dependent manner with Cd concentration.Exogenous cysteine treatment enhanced H₂S level and maintained H₂S at high level under Cd stress,and exogenous H₂S had a similar effect on endogenous cysteine.H₂S reduced the bioavailability of Cd mainly by inhibiting the absorption of Cd and promoting the formation of Cd S nanoparticles,which suggested that cysteine and its metabolite H₂S play an important role in response to Cd stress in T.thermophila.Overall,two cysteine synthesis pathways were found in the free-living protozoa for the first time:de novo pathway and reverse transsulfuration pathway.The cysteine synthesis pathway of T.thermophila exists in different compartments.These enzymes catalyze different substrates.Furthermore,H₂S,the metabolite of cysteine,alleviated Cd stress mainly through inhibiting Cd absorbtion,promoting Cd S nanoparticles formation,increasing oxidation resistance,and regulation of transport in T.thermophila.These findings will expand our understanding for sulfur-containing amino acids metabolism in unicellular free-living protists,and provided new insights for evolutionary pathways of cysteine synthesis and adaptive molecular mechanisms in environmental heavy metal stress.