Comparative Transcriptome Analysis Uncovers Roles of Hydrogen Sulfide for Alleviating Cadmium Toxicity in Tetrahymena Thermophila

doi:10.21203/rs.3.rs-24761/v1

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Comparative Transcriptome Analysis Uncovers Roles of Hydrogen Sulfide for Alleviating Cadmium Toxicity in Tetrahymena Thermophila

https://doi.org/10.21203/rs.3.rs-24761/v1

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published 06 Jan, 2021

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Background

Cadmium (Cd) is a nonessential heavy metal with potentially deleterious effects on different organisms. The organisms have evolved sophisticated defense system to alleviate heavy metal toxicity. Hydrogen sulfide (H₂S) effectively alleviates heavy metal toxicity in plants. However, the function of H₂S for alleviating heavy metal toxicity in aquatic organisms remains less clear. Tetrahymena thermophila is an important model organism to evaluate toxic contaminants in an aquatic environment. In this study, the molecular roles of exogenously H₂S application were explored by RNA sequencing under Cd stress in T. thermophila.

Results

The exposure of 30 µM Cd resulted in T. thermophila growth inhibition, cell nigrescence, and malondialdehyde (MDA) content considerably increase. However, exogenous H₂S (70 µM) significantly alleviated the Cd-induced toxicity and improved antioxidant system. 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 H₂S treatment under Cd stress (1087 upregulated and 272 downregulated). 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 H₂S 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 H₂S treatment under Cd stress in T. thermophila. The relative expression levels of six DEGs were further confirmed through quantitative real-time polymerase chain reaction (qRT-PCR).

Conclusion

H₂S alleviated Cd stress mainly through increasing oxidation resistance, enhancing detoxification, and regulation of transport in free-living unicellular T. thermophila. These findings will expand our understanding for H₂S functions in the freshwater protozoa.

Epigenetics & Genomics

Tetrahymenathermophila

Cd stress

H2S

Transcriptome

Oxidation resistance

Detoxification

Regulation of transport

Heavy metal contamination in aquatic environments has become a global issue [1]. Heavy metals cause adverse effects on the environment because of their toxicity, persistency, and nonbiodegradability [2]. Cadmium (Cd) is one of the most deleterious and serious environmental pollutant to animals and plants [3]. It directly disturbs protein structures and inhibits enzyme activities and causes the formation of reactive oxygen species (ROS), such as superoxide anion (O^•₂⁻), hydroxyl radical (OH^•), and hydrogen peroxide (H₂O₂), which in turn induce oxidative stress and membrane damage [4]. ROS leads to serious damages to different macromolecules, such as DNA, RNA, proteins, and lipids [5]. To survive against the stresses, different organisms have evolved a complex of mechanisms involving multiple genes and strategies at physiological, molecular and metabolic levels, such as activating antioxidants, increasing efflux, and overexpressing metal chelators. The organisms effectively respond to ROS through enzymatic and nonenzymatic antioxidant systems [6]. Superoxide dismutase (SOD) is responsible for the conversion of superoxide radicals to H₂O₂. Catalase (CAT) decomposes H₂O₂ into H₂O and O₂ [7]. Glutathione (GSH) directly or indirectly protects against ROS-mediated cell injury. Several GSH-associated enzymes, such as glutathione reductase, glutathione peroxidase (GPX), and glutathione S-transferase (GST), cumulatively protect against ROS under toxic metal stress [8]. Recent studies revealed that exogenous gaseous signal molecule hydrogen sulfide (H₂S) improve Cd tolerance in plants by reducing oxidative damage.

H₂S is produced endogenously from cysteine mainly by cystathionine β-synthase and cystathionine γ-lyase and is important for various physiological functions in mammals, including synaptic transmission, vascular tone, inflammation, angiogenesis, and protection from oxidative stress [9]. Exogenous H₂S acts as a potent antioxidant under Cd stress by enhancing antioxidant enzymes activities in wheat seedlings [10], and alleviates Cd toxicity through regulations of Cd transport across the plasma and vacuolar membranes in Populus euphratica cells [11]. In Brassica rapa, H₂S mitigates Cd-induced cell death by inhibiting ROS accumulation [12]. H₂S reduced Cd-induced oxidative stress, particularly by enhancing redox status and the activities of ROS and methylglyoxal detoxifying enzymes in rice [13]. However, the functions and signal pathways of H₂S under heavy metal stress remain unclear in other organisms.

Ciliates are highly divergent unicellular eukaryotic organisms with nuclear dualism. These unicellular eukaryotic organisms are ubiquitous in various environments [14]. Ciliates play an important role in aquatic ecosystem and are used as whole cell biosensors to evaluate toxicity of various environmental pollutants [15, 16]. Tetrahymena thermophila is a free-living ciliate widely distributed in freshwater environment. It is an excellent model organism for toxicological and ecotoxicological studies in aquatic toxicity test systems. T. thermophila contains a large number of gene families that are involved in processes associated with sensing and responding to environmental stresses. The 44 P450 monooxygenase genes and 165 ATP-binding cassette (ABC) transporter genes were identified in T. thermophila [17, 18]. The 70 putative GST genes exist in the macronuclear genome of T. thermophila, which imply that this organism has been exposed to diverse xenobiotics throughout its evolution [19]. T. thermophila has higher sensitivity to heavy metal stress [20]. One of the basic heavy metal resistance mechanisms present in T. thermophila is the intracellular sequestration (bio-accumulation), of which the cellular detoxification processes is the chelation of metal cations by endogenous proteins or peptides, such as metallothioneins (MTs), phytochelatins (PCs), and GSH [14]. Heavy metal stress responsive genes and antioxidant defense system allow the survival of Tetmemena in presence of metals in the environment [21]. These heavy metal stress response mechanisms in freshwater ciliates are kinds of self-protection through intracellular regulation.

However, little is known about the mitigative effects on heavy metal stress through exogenous additive in the freshwater protozoa. In this study, we found H₂S promoted T. thermophila proliferation and alleviated cellular toxicity induced by Cd. The mechanism of H₂S function on T. thermophila stressed under Cd was evaluated by high throughput transcriptome sequencing technology. These findings will expand our understanding for H₂S functions in aquatic organisms.

H₂S mitigates inhibition of proliferation of T. thermophila under Cd stress

Heavy metal pollutants caused toxic effects on ciliates, and the effect varied according to the bioavailable concentration and nature of the heavy metal [22]. An assay using the motile response of Tetrahymena pyriformis, gave a sensitivity better than 1 µM and a toxicity threshold to 7 µM for Cd [23]. Cd caused a dose-dependent decline in the viability of T. thermophila [24]. To understand the tolerance level of Cd for T. thermophila, the half maximal inhibitory concentration (IC50) value of Cd was determined, and it was calculated to be 30 µM for T. thermophila cells at 6 h culture (Fig. 1a). H₂S alleviates Cd toxicity in plants. Exogenous H₂S recovered Cd-induced growth inhibition in Brassica napus, Arabidopsis, and barley [25–27]. 70 µM H₂S largely stimulated proliferation of T. thermophila (Fig. 1b). Furthermore, the proliferation of T. thermophila under Cd and H₂S treatments was investigated to explore whether H₂S physiologically alleviate Cd toxicity. An amount of 0.7 × 10⁵ mL^− 1 cells was transferred to the SPP medium, and the number of cells was counted every 4 h. The proliferation inhibition of T. thermophila under 30 µM Cd was dramatically mitigated by H₂S (Fig. 1c). The results showed exogenous H₂S play a protective role on Cd stress in T. thermophila.

H₂S alleviates lipid peroxidation and improves antioxidant capacity under Cd stress

Cd significantly inhibits the growth of microorganisms and plants. The treatment using 30 µM Cd also led to the stunted growth and nigrescence of T. thermophila after being exposed for 24 h. However, the toxic symptoms were drastically alleviated with H₂S supplement. The exogenous H₂S significantly inhibited Cd accumulation (decrease by 26%) in the T. thermophila cells (Fig. 2a).

Cd stress caused lipid peroxidation and induced malondialdehyde (MDA) production of T. thermophila cells in a dose-dependent manner (Fig. 2b). Low concentrations of Cd had less effect on the MDA content in T. thermophila cells. However, 30 and 50 µM Cd lead to the MDA content of the cells increased by 91% and 395%, respectively. In comparison, the MDA content of the cells had no significant changes when the cells were treated with H₂S. However, the H₂S treatment markedly decreased the MDA content of T. thermophila cells under Cd stress (Fig. 2c).

It is well known that antioxidant defense system increases organism tolerance against metal-induced toxicity by upregulating the nonenzymatic antioxidants and different antioxidant enzymes. H₂S increase GSH content and antioxidant enzymes activity in Arabidopsis [26]. Under Cd stress, GSH content increased by 51% and exogenous H₂S supplement further increased the GSH content in T. thermophila (Fig. 2d). Furthermore, SOD activity increased by 92% and CAT activity increased by 29% under Cd stress. H₂S supplement also enhanced SOD and CAT activities in T. thermophila cells (Fig. 2e, f). The results indicated that H₂S could alleviate Cd toxicity by improving the antioxidant capacity of T. thermophila cells.

Characterization Of The Sequenced Illumina Libraries

Tetrahymena evolved various efficient detoxification pathways allowing the survival from Cd stress, such as overexpressing metal chelators and activating antioxidant signal pathways. To explore the mechanisms of H₂S alleviating Cd stress in T. thermophila, RNA-seq was employed to investigate the changes in genome-wide gene expression for four groups of cells: exposed to nutrients only (CK), with 70 µM NaHS (S), under 30 µM CdCl₂ (C), and 70 µM NaHS + 30 µM CdCl₂ (CS), with three biological replicates. A total of 305.2 million pair-end reads with Q20 > 97% and Q30 > 93% were obtained from 12 libraries (Table 1). Among the short clean reads, more than 94% were mapped to the T. thermophila Functional Genomics Database (http://tfgd.ihb.ac.cn/). Approximately 95% of the reads from each library were perfectly matched to the reference genes, and more than 93% of the reads in the libraries were mapped to single locations. Half of these uniquely mapped reads in each library were mapped to the sense strand, whereas the other half was mapped to the antisense strand (Additional file 1: Table S1). Then, the mapped reads were further classified and annotated using TopHat [28]. The correlations between the three replicated samples were calculated on the basis of the normalized expression results (Additional file 2: Figure S1). The correlation coefficient between the three replicated samples was reasonable for the CK, S, and CS groups, but that between C1 and C2 or between C1 and C3 was lower than 70%. Thus, the C1 sample was abnegated.

Differentially expressed genes (DEGs) in response to H₂S treatment, Cd stress, and H₂S treatment under Cd stress

DEGs were hierarchically clustered to obtain a comprehensive view of the differential gene expression under H₂S treatment, Cd stress, and H₂S treatment with Cd stress (Fig. 3a). Under H₂S treatment, the expression level of 191 genes changed. Among them, 134 genes were upregulated and 57 genes were downregulated. Under Cd stress, the expression level of 9152 genes significantly changed, including 4658 upregulated genes and 4494 downregulated genes. A total of 1087 genes were upregulated and 272 genes were downregulated with H₂S treatment under Cd stress. The expression levels of most genes recovered under Cd stress with H₂S treatment. A total of 4122 genes were upregulated and 3738 genes were downregulated between Cd stress and H₂S treatment under Cd stress (Fig. 3b).

The 95 DEGs overlapped between CK vs. S (50.0%) and C vs. CS (1.2%), indicating that H₂S-responsive transcripts under normal condition were far fewer than those under Cd stress. The 806 DEGs in CK vs. C (8.8%) were common to CK vs. CS (59.3%), and the 6001 genes overlapped between CK vs. C (65.6%) and C vs. CS (76.3%) (Fig. 3c), suggesting most of the Cd-responsive transcripts were altered by H₂S. The absolute value of the log2 ratio ranged from 1.00 to 14.70 in CK vs. C, and ranged from 1.00 to 12.21 in C vs. CS (Fig. 3d). The significantly upregulated genes under Cd stress (log2FC > 8) were considerably related to oxidoreductase, GPXs, GSTs, heat shock protein, and MTs (Additional file 3: Table S2). Compared with Cd stress, the unigenes significantly downregulated in the H₂S treatment under Cd stress (log2FC < -8) were mainly related to oxidoreductase, GPXs, GSTs, and heat shock protein (Additional file 4: Table S3). The results indicated that the redox system is sensitive for H₂S treatment and Cd stress in T. thermophila.

Gene Ontology (go) Enrichment Analysis Of Degs

To obtain the functional annotations of the DEGs for Cd stress and H₂S treatment under Cd stress, GO category enrichment analysis was performed. For the comparison of CK vs. C, the 5740 DEGs were classified as 50 functional groups (Fig. S2). The functional groups were divided into three categories: biological process, molecular function, and cellular component. The biological process mainly comprises DEGs involved in metabolic process (2619, 45.63%), cellular process (2589, 45.10%), single-organism process (1406, 24.49%), biological regulation (777, 13.5%), and localization (706, 12.30%). In the category of cellular components, membrane (2711, 47.23%), membrane part (2517, 43.85%), cell (1798, 31.32%), cell part (1784, 31.08%), and organelle (1094, 19.06%) were the most represented groups. Among the molecular function category, the major groups were catalytic activity (3081, 53.68%), binding (2160, 37.63%), and transporter activity (416, 7.25%). For the comparison of C vs. CS, the 4883 DEGs were also classified as 50 functional groups. Between C vs. CS and CK vs. C, they had exactly similar classification patterns (Additional file 5: Figure S2).

Next, TopGO enrichment analysis was performed to obtain a detailed classification through false discovery rate (FDR) adjusted P-value of < 0.05 as the cutoff (Fig. 4). The distribution of enriched GO terms indicated that several DEGs were involved in oxidation–reduction process (GO:0055114), oxidoreductase activity (GO:0016491) and glutathione peroxidase activity (GO:0004602) in both CK vs. C and C vs. CS. Under Cd stress, 283 DEGs were included in oxidoreductase activity and 231 DEGs participated in oxidation-reduction process. Compared with Cd stress, 263 DEGs constituted the oxidoreductase activity with H₂S addition, and 182 DEGs involved in oxidation–reduction process. Furthermore, response to oxidative stress (GO:0006979) was enriched in CK vs. C. Cell redox homeostasis (GO:0045454) was enriched in C vs. CS. These data indicated that H₂S responds to Cd stress mainly through the adjustment of the redox balance.

Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway enrichment analysis

The annotated T. thermophila transcripts were mapped to the KEGG pathways to investigate the genes involved in important metabolic pathways. Under Cd stress, the 1116 DEGs were mapped to the 252 KEGG pathways. For H₂S treatment under Cd stress compared with Cd stress, 966 DEGs were mapped to 247 KEGG pathways. The pathways considerably related to carbon metabolism, GSH metabolism, drug metabolism–cytochrome P450, and metabolism of xenobiotics by cytochrome P450 (Fig. 5a, b). Under Cd stress, 54 DEGs (4.84%) were distributed in the carbon metabolism, and 48 DEGs (4.97%) also enriched in this pathway with adding H₂S. The KEGG pathway of GSH metabolism includes primarily GPX and GST. 52 DEGs (4.66%) were enriched at the GSH metabolism under Cd stress, and 54 DEGs (5.59%) were also enriched with adding H₂S. Under Cd stress, 39 DEGs (3.49%) or 40 DEGs (3.58%) were distributed in drug metabolism–cytochrome P450 or metabolism of xenobiotics by cytochrome P450, and 40 DEGs (4.14%) or 42 DEGs (4.35%) were enriched in the two pathways with H₂S addition, respectively. Besides, under Cd stress, 67 DEGs (6.00%) were distributed in ABC transporters (Additional file 6: Figure S3), and 70 DEGs (7.25%) were enriched in this pathway with adding H₂S. The considerably enriched pathways in C vs. CS were similar to those of CK vs. C, indicating the important regulatory mechanisms through H₂S under Cd stress.

Regulation of cytochrome P450 and GSH metabolism under Cd stress and with H ₂S treatment

Cytochrome P450 represents an important participant in regulatory networks of organism responses to Cd stress. The expression of genes encoding cytochrome P450 family proteins was strongly induced by Cd in rice [29]. In the wolf spider Pardosa pseudoannulata, cytochrome P450 genes were found to respond to Cd stress [30]. A total of 44 putative functional cytochrome P450 genes were identified and classified into 13 families and 21 sub-families according to standard nomenclature in T. thermophila. Cd induced the expression of 6 cytochrome P450 genes and inhibited the expression of 9 cytochrome P450 genes (|Log2FC|>1). H₂S addition reversed the expression of 11 of them (Table 2). Under Cd stress, metabolism of xenobiotics by cytochrome P450 was significantly enriched by KEGG pathway analysis on DEGs, of which 32 DEGs were markedly upregulated (Additional file 7: Figure S4), including 30 GSTs, and 29 of them were markedly downregulated when H₂S was added.

63 cytosolic GSTs have been identified in T. thermophila. Under Cd stress, GSH metabolism was significantly enriched by KEGG pathway analysis on upregulated genes (Fig. 6). The upregulated DEGs included 31 GSTs and 8 GPXs. Compared with Cd stress, GSH metabolism was also significantly enriched on downregulated genes with H₂S addition, including 30 GSTs and 8 GPXs. Specifically, the 8 upregulated GPXs and 29 GSTs induced by Cd stress returned to normal level with adding H₂S. GSTs detoxify xenobiotic compounds by linking the -SH group of antioxidant GSH covalently to a substrate [31]. Given the role of GSH in the detoxification of heavy metals, regulation of GSH, GPXs, and GSTs by H₂S play an important role in ameliorating Cd stress.

Regulation of ABC transporters under Cd stress and with H₂S treatment

GSH marked xenobiotic molecules are excreted from cells via phase III proteins such as ABC transporter enzymes. Based on the KEGG pathway analysis, the ABC transporters related to detoxification of Cd in T. thermophila was screened and listed in Fig. 7. Under Cd stress, 49 DEGs involved in the enrichment of ABC transporters were markedly upregulated, while 18 DEGs were downregulated (Fig. 7). The ABC transporter family of T. thermophila was classified into 8 distinct groups [18]. The ABCA family comprises of a total of 32 members, which included 9 upregulated genes and 3 downregulated genes under Cd stress. The ABCB family consists of 26 transporters, of which 5 genes were upregulated and 5 genes were downregulated. The ABCC family comprises 60 transporters and represents the largest family of ABC transporters in T. thermophila. 22 genes were induced by Cd and 6 genes were downregulated. The ABCG family includes 39 transporters, and 9 genes were upregulated under Cd stress and 3 genes were inhibited. ABCG19 (TTHERM_00034920) was induced up to 357 folds under Cd stress (Additional file 3: Table S2), which suggested it could play an important role in detoxifying Cd toxicity. H₂S has been demonstrated for direct regulating ABC transporters to induce stomatal movement in plants. Exogenous H₂S induces stomatal closure and this effect is impaired by the ABC transporter inhibitor glibenclamide in guard cells [32]. Compared with Cd stress, 42 DEGs were downregulated and 28 DEGs were upregulated for ABC transporters with H₂S addition. Most of the expression of ABC transporters under Cd stress was restored by exogenous H₂S addition.

Quantitative real-time polymerase chain reaction (qRT-PCR) validation of differentially expressed transcripts

To validate the accuracy and reliability of transcriptome sequencing, six DEGs involved in chelating heavy metals, redox reaction, and stress response were evaluated through qRT-PCR. H₂S slightly affected the expression of the genes under non-stressed condition. Under Cd stress, the expression levels of the genes were significantly upregulated. The log2FC of the CdMT genes were ranked as MTT5 ≈ MTT3 > MTT1 under Cd stress (Additional file 3: Table S2). The expression levels of four genes decreased with H₂S addition, including SSA6, OXR1, GLR2, and MTT3. On the contrary, the expression level of MTT1 further increased and MTT5 had no obvious change with H₂S treatment under Cd stress (Fig. 8). The three CdMT isoform genes present differential expression patterns between Cd stress and H₂S treatment under Cd stress, indicating functional diversification. The correlation between qRT-PCR and RNA-seq was measured by scatter plotting log2-fold changes, which showed a positive correlation coefficient in both techniques (Pearson coefficient R² = 0.95), thereby indicating the reliability of the sequencing data.

H₂S treatment improved Cd tolerance inT. thermophila

Cd is a nonessential and highly toxic metal that adversely affects the growth and development of different living organisms. 50–150 µM Cd inhibited the elongation of Arabidopsis roots in a dose-dependent manner [26]. Survival rate of three ciliates decreased remarkably with increasing Cd exposure, and the LC50 for Notohymena (5 µg/mL) was higher than Tetmemena (2 µg/mL) and Euplotes (2 µg/mL) [33]. The LC50 of Cd for T. thermophila SB1969 strain was 44.5 µM for 24 h exposure [34]. In the present study, IC50 of Cd for T. thermophila was 30 µM for 6 h treatment. Several strategies have been developed to alleviate the toxicity of Cd. For example, H₂S participate in suppressing Cd stress. 100 µM H₂S supplementation exhibited an inhibitory effect on Cd uptake and accumulation in rice [13]. 300 µM H₂S alleviates Cd toxicity by changing Cd chemical forms and increasing the activities of antioxidant enzymes in salix [35]. We found that 70 µM H₂S promoted the proliferation of T. thermophila and alleviated Cd toxicity by inhibiting the accumulation of Cd in cells (Fig. 1, 2a).

H₂S participate in oxidative damage scavenging systems inT. thermophila

The antioxidant systems of enzymatic and nonenzymatic antioxidants could immediately abolish ROS. H₂S alleviates oxidative damage against copper stress in wheat, and toxic effects of lead on cotton antioxidant activity [36, 37]. In ROS scavenging pathways, O₂⁻ is converted to H₂O₂ by SOD, and H₂O₂ is subsequently reduced to H₂O and O₂ by CAT. 30 µM Cd induced a sharp generation of MDA and enhanced activity of SOD and CAT in T. thermophila (Fig. 2). The SOD and CAT enhance the basal antioxidant capacity to overcome oxidative stress caused by Cd [38]. GSH acts as substrate for GPX to catalyze H₂O₂ [33]. The GSH content was notable upregulated under Cd stress, and with the addition of H₂S, the GSH content further increased (Fig. 2). The improvement of GSH levels and increase of SOD and CAT activities under H₂S treatment promoted detoxification of ROS, and protected the cells from oxidative damage. In addition, the accumulation of MDA content significantly reduced by the exogenous H₂S treatment. The exogenous H₂S indirectly balance the O₂⁻ and H₂O₂ levels and maintain redox balance in T. thermophila.

Cd stress leads to the significant molecular and physiological changes in different organisms. The whole transcriptional reprogramming is regarded as a vital molecular response to Cd stress. Enzymes responsible for detoxification and antioxidant process can alleviate oxidative stress. GPX protects organisms from oxidative damage by catalyzing the reaction between ROS and GSH [39]. Under arsenate stress, glutathione peroxidase family protein was over-expressed in T. pyriformis, which is involved in protecting the cells from oxidative damage [40]. Oxidoreductases are enzymes that participate in the oxidation-reduction process, and Cd exposure led to the remarkable variations of these genes in crayfish hepatopancreas [41]. Under Cd stress, 27 oxidoreductases and 9 glutathione peroxidase family proteins were upregulated in the oxidation–reduction process enrichment term in T. thermophila through GO analysis. Among them, the expression levels of 25 oxidoreductases and the all 9 glutathione peroxidase family proteins returned to normal upon the H₂S addition. The redox system was disrupted after Cd exposure and recovered through H₂S supplement in T. thermophila.

MTs are sulfhydryl-rich proteins, which have been known as multifunctional proteins and as pivotal members of the cellular integrated stress response system. T. thermophila has three CdMTs (MTT1, MTT3, and MTT5) and two CuMTs (MTT2 and MTT4). MTT5 is the strongest induced, while MTT3 is the least induced by Cd stress at 1 h or 24 h [34, 42]. However, the ranking of relative expression values can change relying on the duration of heavy metal treatment [14]. We found that the ranking of induction fold change values was MTT5 ≈ MTT3 > MTT1 when cells was stressed by Cd stress at 6 h (Fig. 6, and Additional file 3: Table S2). MTs also display oxyradical scavenging capacity and specifically neutralize hydroxyl radicals [43]. Interestingly, the upregulated expression of MTT1 under Cd stress further increased with H₂S treatment. On the contrary, the upregulated expression of MTT3 under Cd stress decreased with H₂S treatment. Meanwhile, the upregulated expression of MTT5 under Cd stress has no significant change with H₂S treatment. These data further indicated that different MT genes have conserved and various function.

To protect the cell from oxidative stress, heat shock proteins (HSPs) are greatly induced. In response to Cd exposure for 24 h in Tetmemena, there was around 46-fold increase in the hsp70 transcriptional expression as compared to control [21]. In the present study, three HSPs (TTHERM_000351139, TTHERM_00351140, TTHERM_00171850) were found to activate by Cd and the expression level returns to normal by H₂S addition ( Fig. 6, and Additional file 3: Table S2). So, H₂S involved in the detoxification of Cd through the antioxidant signal pathway.

H₂S alleviates Cd stress by regulating cytochrome P450, GST and ABC transporters

The metabolism of xenobiotics comprises of three phases, including modification (phase I), conjugation (phase II), and excretion (phase III). Toxicity responsive genes of T. thermophila has been partially revealed, including cytochrome P450 gene family in phase I, GST gene family in phase II, and the ABC transporter superfamily in phase III [19]. Cytochrome P450 is in charge of the metabolism of most xenobiotics and efficiently eliminates foreign chemicals [30]. Cytochrome P450 was induced at 6 h of Cd treatment, then decreased to control levels after 18–24 h, and expressed at low level after 48 h of Cd exposure in Physcia adscendens [44]. We found that Cd altered the expression of 15 cytochrome P450 genes, and H₂S reversed the expression of most of these genes in T. thermophila.

P450 enzymes and GSTs are a well-conserved mechanism of the Cd response in a wide range of organisms [29]. GSTs are a large protein family with a well-established role in the conjugation of xenobiotics with GSH [45]. GSH form complexes with heavy metals and serves as the first line of defense against heavy metals stress [46]. GST plays important roles through GSH metabolism in the protection against oxidative damage resulting from the high levels of ROS under Cd stress in Chironomus riparius [47]. In Euplotes crassus, copper and zinc significantly increased the expression of GST [48]. In animals, Cd is a redox-inactive metal that binds to the thiol group of GSH [49]. The expression level of 31 GSTs was upregulated under Cd stress. Among of them, the expression level of 29 GSTs was downregulated with H₂S addition in T. thermophila. The results showed that H₂S alleviates Cd stress through glutathione metabolism.

GSH marked xenobiotic molecules are transported via phase III proteins such as ABC transporters. The ABC transporters comprise large protein families that mediate various physiological processes [50]. In fission yeast, Cd forms complexes with either GSH or PCs and subsequently transports into vacuoles via the ABC transporters. The ABC transporters in plants transport GS₂–Cd or PC–Cd complexes into subcellular compartments or out of cells [51]. MRP1 is a well-known ABCC family gene in human, and also regarded as GS-X pumps [52]. Moreover, MRP5, another ABC transporter C family gene, has an important role in the detoxification of Cd [53]. MRP proteins are potential mediators of heavy metal resistance in zebrafish cells [54]. ABCC52 is a MRP-like gene in T. thermophila and induced by Cd and recoverd through H₂S treatment. In T. thermophila, the membrane pump protein encoded by ABCT gene abcb15 can enhance the tolerance of DDT and protect cells from this exogenous toxin by efficiently pumping the toxin to the extracellular space [55]. The ABCB15 of T. thermophila shares a 32.3% amino acid identity with ABCB1 in human, which has been shown to confer resistance to or transport a wide variety of toxic compounds [18]. The induction of ABCB1 was detected in Euglena exposed to Cd [56]. Exposure to Cd in the gills and hepatopancreas of oyster showed activation of ABCB1 protein but no significant changes to its mRNA, suggesting post-transcriptional regulation of ABCB1 [57]. We do not detect the change of ABCB15 at transcriptional level under Cd stress in T. thermophila, and the post-transcriptional regulation needs further study. As a whole, a total of 67 DEGs were enriched in the ABC transporter pathway under Cd stress, and the ABC transporter pathway was significantly altered with H₂S addition. So, the decrease of Cd accumulation and toxicity under H₂S treatment partially attribute to regulation of Cd transport by ABC transporter pathway in T. thermophila cells.

H₂S physiologically alleviated growth inhibition under Cd stress and accumulation of Cd in T. thermophila. It mitigated lipid peroxidation and improved GSH levels as well as SOD and CAT activities for detoxification of ROS. H₂S affected various metabolic processes and molecular functions under Cd stress, including glutathione metabolism, metabolism of xenobiotics by cytochrome P450, GPXs, Cytochrome P450s, GSTs, and ABC transporters (Fig. 7). H₂S protects T. thermophila from Cd stress by increasing oxidation resistance, enhancing detoxification, and regulation of transport.

Strains and culture conditions

T. thermophila B2086 was obtained from the Tetrahymena Stock Center (http://tetrahymena.vet.cornell.edu/, Cornell University, Ithaca). The cells were cultured in a 1 × SPP medium (pH 7.4) containing 1% proteose peptone, 0.1% glucose, 0.2% yeast extract, and 33.3 µM EDTA-FeNa₂ at 30 °C with continuous shaking [58]. For physiological and biochemical analyses, cells were transferred to 10 mL of SPP medium with the same initial concentration at 0.7 × 10⁵ mL^− 1.

Cellular Proliferation Analysis

Various Cd concentrations ranging from 0.5 µM to 256 µM were used to determine the IC50 of Cd for T. thermophila cells for 6 h treatment. The S-curve was plotted with GraphPad Prism 5 in terms of inhibition rates and Cd concentrations. In a pilot study, 6 levels of NaHS (the H₂S donor) concentrations (0, 50, 70, 100, 200, 400 µM) were explored for treating T. thermophila cells. Based on the results of the pilot study, 70 µM NaHS was identified as the optimum positive effect H₂S concentration, which was subsequently used as the processing condition in the subsequent experiment (Fig. 1b). To analyze the effects on cell proliferation of H₂S under Cd stress, cells were uniformly grown in the SPP medium with 30 µM Cd (IC50) in the presence or absence of 70 µM NaHS (optimum positive effect concentration), and their starting concentration was 0.7 × 10⁵ mL^− 1. Each treatment was replicated 3 times, and the sample was taken every 4 h. The number of cells was determined using a blood cell counting chamber. The formation of CdS by NaHS and CdCl₂ was negligible at present low Cd and H₂S concentrations [35].

Measurement Of Cd Concentration

T. thermophila cells were cultured with 30 µM Cd alone or with the addition of 70 µM NaHS treatments for 24 h. After centrifugation, the cells that accumulated Cd were removed from the medium. The Cd in the medium was measured using inductively coupled plasma atomic emission spectroscopy (ICP-OES, iCAP 7000 Series, Thermo Scientific). The results represented the average of three independent experiments.

Assay Of Mda, Gsh, Sod, And Cat

The cells (3 × 10⁶) were exposed to various Cd concentrations for 6 h or 70 µM NaHS before they were collected and ultrasonically lysed. The lysate was centrifuged at 12,000 g for 20 min at 4 °C. The protein quantitation was evaluated through the bicinchoninic acid (BCA) assay. The amount of lipid peroxidation product MDA was determined by the thiobarbituric acid (TBA) method according to the reaction of MDA with TBA [59]. Each experiment was done in triplicate.

The cells in the logarithmic phase were divided into four groups with different treatments: (1) CK, without chemical treatments (the standard SPP solution); (2) S, treatment with NaHS (70 µM) and no Cd; (3) C, treatment with Cd (30 µM) and no NaHS; and (4) CS, treatment with Cd (30 µM) and NaHS (70 µM). After treatment for 6 h, the cells were centrifuged and ultrasonically lysed. The antioxidant contents of GSH were determined by reacting with 5, 5′-dithiobis-(2-nitrobenzoic acid) (DTNB), and the yellow products were evaluated by colorimetric method at 405 nm according to the instruction in the GSH assay kit (Nanjing Jiancheng Bioengineering Institute, china). A microtiter plate assay was used to determine the SOD activities utilizing a water-soluble tetrazolium salt (WST-1), which was used as a detector of superoxide radical generated by xanthine oxidase and hypoxanthine [60]. The SOD activity was measured with the standard SOD assay kit (Nanjing Jiancheng Bioengineering Institute, China). One unit of enzymatic activity caused 50% inhibition of WST-1 formazan. CAT activities were determined by Goth’s colorimetric method. The supernatant was incubated with H₂O₂ substrate, and the enzymatic reaction stopped by the addition of ammonium molybdate. The intensity of the yellow complex was formed by molybdate and measured at 405 nm [61]. One unit of activity indicated 1 µmol of decomposition of H₂O₂ per second. Each experiment was repeated thrice.

Extraction Of Total Rna

A total of 3 × 10⁶ cells were harvested from the CK, S, C, CS samples with 3 replications, immediately frozen in liquid nitrogen, and stored at − 80 °C. Total RNA was manually extracted using a TRIzol regent (Invitrogen, USA) in accordance with the manufacturer’s instructions. RNA contamination and degradation were monitored on 1% agarose gels. RNA purity was checked with a NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). RNA concentration was measured with a Qubit® RNA assay kit in a Qubit®2.0 fluorometer (Life Technologies, CA, USA). RNA integrity was evaluated using an RNA Nano 6000 assay kit of an Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).

Library Construction, Illumina Sequencing, And Transcriptome Assembly

A total amount of 3 µg RNA was used as input material for the RNA sample preparations. Sequencing libraries were generated using a NEBNext®Ultra™ RNA library prep kit for Illumina® (NEB, USA) in accordance with the manufacturer’s instructions, and index codes were added to attribute the sequences to each sample. mRNA was purified from the total RNA using poly-T oligo-attached magnetic beads. Fragmentation was conducted using divalent cations at an increased temperature in a NEBNext first-strand synthesis reaction buffer (5×). First-strand cDNA was synthesized using a random hexamer primer and M-MuLV reverse transcriptase (RNase H). Second-strand cDNA was subsequently synthesized using DNA polymerase I and RNase H. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After the 3′ ends of the DNA fragments were adenylated, a NEBNext adaptor with a hairpin loop structure was ligated to prepare for hybridization. The library fragments were purified with an AMPure XP system (Beckman Coulter, Beverly, USA) to select the cDNA fragments with preferentially 150–200 bp in length. Then, 3 µL of USER Enzyme (NEB, USA) was used with a size-selected, adaptor-ligated cDNA initially at 37 °C for 15 min and then at 95 °C for 5 min. Then, PCR was performed with Phusion high-fidelity DNA polymerase, universal PCR primers, and index (X) primer. PCR products were purified (AMPure XP system), and library quality was assessed using the Agilent Bioanalyzer 2100 system. The index-coded samples were clustered on a cBot cluster generation system using a TruSeq PE cluster kit v3-cBot-HS (Illumina) in accordance with the manufacturer’s instructions. After generating the cluster, the prepared libraries were sequenced on an Illumina Hiseq 2000 platformto generate paired-end reads. Transcriptome assembly was accomplished using Trinity [62] with min_kmer_cov set to 2 by default. All of the other parameters were set to default.

Gene Expression Level Quantification And Differential Expression Analysis

Gene expression levels were estimated by RSEM [63] for each sample. Clean data were mapped on the assembled transcriptome, and the read count for each gene was obtained from the mapping results. Two conditions/groups were subjected to differential expression analysis using the DESeq R package (1.10.1). The resulting P values were adjusted using Benjamini and Hochberg’s approach for controlling the FDR. Genes with an adjusted P < 0.05 and |log2 (fold-change)| > 1 found by DESeq were assigned as differentially expressed. The GO enrichment analysis of DEGs was implemented by the topGO R packages in a Kolmogorov–Smirnov (KS) test. The KEGG [64] was used to understand the functions of cells at the molecular-level (http://www.genome.jp/kegg/). KOBAS was used to evaluate the statistical enrichment of DEGs in KEGG pathways [65].

Qrt-pcr Validation

The total RNAs of the four different groups were extracted from the frozen cell samples with RNAiso Plus (TaKaRa, Japan). Total RNA (2 µg) was reverse-transcribed into first-strand cDNA using PrimeScript™ RT reagent kit with gDNA Eraser (Takara, Japan). qRT-PCR was conducted using an SYBR Green Ⅱ (SYBR® Premix Ex Taq™ Kit, Takara) in a CFX96TM real-time system (BIO-RAD, USA) through the following steps: heat for 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C and extension for 30 s at 60 °C. A melting curve analysis was performed after every PCR reaction to confirm the accuracy of each amplified product. The 17S rRNA gene was used as an internal control. All primers used for qRT-PCR are listed in Additional file 8: Table S4. Amplification efficiency (E) was measured by using 4-fold serial dilutions of a positive control PCR template. The efficiency requirement was met for all the tested genes. Results were finally processed by the standard-curve method [66]. Their amplification efficiencies were greater than 90% and the correlation coefficients (R²) were all 99% (Additional file 9: Table S5). The relative expression levels of genes were calculated using the 2^−ΔΔCT method [67]. Three independent replicates were performed for each treatment. The correlation analysis of qRT-PCR and RNA-seq were based on Pearson’s correlation coefficient.

Statistical analysis

Each treatment was conducted in three replicates. Data were subjected to an analysis of variance (ANOVA). Duncan’s multiple range test was used to compare significant differences at P < 0.05 among various treatments. Different letters indicated significantly different results.

Cd: Cadmium; H₂S: Hydrogen sulfide; NaHS: Sodium hydrosulfide; T. thermophila: Tetrahymena thermophila; ROS: Reactive oxygen species; O^•₂⁻: Superoxide anion; OH^•: Hydroxyl radical; H₂O₂: Hydrogen peroxide; SOD: Superoxide dismutase; CAT: Catalase; GSH: Glutathione; GPX: Glutathione peroxidase; GST: Glutathione S-transferase; MDA: Malondialdehyde; MTs: metallothioneins; PCs: phytochelatins; IC50: Half maximal inhibitory concentration; CK: Control; S: Control with H₂S treatment; C: Control under Cd stress; CS: Control with H₂S treatment under Cd stress; DEGs: Differentially expression genes; FC: Fold Change; FDR: False discovery rate; FPKM: Fragments per kilo base million; KS: Kolmogorov–Smirnov; GO: Gene ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; qRT-PCR: quantitative real-time polymerase chain reaction; E: Amplification efficiency; R²: Correlation coefficient.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This study was funded by National Natural Science Foundation of China (Grant No. 31872224, 31471999).

Authors' contributions

WW and HL conceived and designed the experiments. HL conducted experiments. HL, WW, JX, and TB analyzed data. HL and WW wrote the manuscript.

Acknowledgements

Not applicable.

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Comparative Transcriptome Analysis Uncovers Roles of Hydrogen Sulfide for Alleviating Cadmium Toxicity in Tetrahymena Thermophila

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Results

H2S mitigates inhibition of proliferation of T. thermophila under Cd stress

H2S alleviates lipid peroxidation and improves antioxidant capacity under Cd stress

Characterization Of The Sequenced Illumina Libraries

Differentially expressed genes (DEGs) in response to H2S treatment, Cd stress, and H2S treatment under Cd stress

Gene Ontology (go) Enrichment Analysis Of Degs

Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway enrichment analysis

Regulation of cytochrome P450 and GSH metabolism under Cd stress and with H 2S treatment

Regulation of ABC transporters under Cd stress and with H2S treatment

Quantitative real-time polymerase chain reaction (qRT-PCR) validation of differentially expressed transcripts

Discussion

H2S treatment improved Cd tolerance inT. thermophila

H2S participate in oxidative damage scavenging systems inT. thermophila

H2S alleviates Cd stress by regulating cytochrome P450, GST and ABC transporters

Conclusion

Methods

Strains and culture conditions

Cellular Proliferation Analysis

Measurement Of Cd Concentration

Assay Of Mda, Gsh, Sod, And Cat

Extraction Of Total Rna

Library Construction, Illumina Sequencing, And Transcriptome Assembly

Gene Expression Level Quantification And Differential Expression Analysis

Qrt-pcr Validation

Statistical analysis

Abbreviations

Declarations

Ethics approval and consent to participate

Consent for publication

Availability of data and materials

Competing interests

Funding

Authors' contributions

Acknowledgements

References

Supplementary Files

Status:

Journal Publication

Version 1

H₂S mitigates inhibition of proliferation of T. thermophila under Cd stress

H₂S alleviates lipid peroxidation and improves antioxidant capacity under Cd stress

Differentially expressed genes (DEGs) in response to H₂S treatment, Cd stress, and H₂S treatment under Cd stress

Regulation of cytochrome P450 and GSH metabolism under Cd stress and with H ₂S treatment

Regulation of ABC transporters under Cd stress and with H₂S treatment

H₂S treatment improved Cd tolerance inT. thermophila

H₂S participate in oxidative damage scavenging systems inT. thermophila

H₂S alleviates Cd stress by regulating cytochrome P450, GST and ABC transporters