1. Metabolomics analysis of iron-rich and iron-deficient cultured T. vaginalis
A total of 4 equal samples from IR and ID trichomonad cells were collected for HPLC-QTOF MS analysis. Generally, principal component analysis (PCA) was performed to show the correlation of the metabolomics dataset among the experimental groups. However, due to the small sample size and multiple dimensions of detected metabolites, PCA is not suitable for our experiment (data not shown). OPLS-DA is another method to measure the similarities and differences between experimental settings (IR and ID). The score plot of OPLS-DA revealed a correlation within IR and ID datasets in positive and negative ion modes (Fig. 1A and B). In addition, permutation tests (n = 999) for each ion mode were performed to further confirm the validity of the two experimental groups (Fig. 1C and D). The R2Y and Q2 values were more than 0.5, suggesting that these clustering trends of major components derived from samples were separated expectedly in both positive and negative ion modes.
A total of 3,277 peaks were present in positive and negative ion modes (1,802 and 1,475, respectively) (Table 1). Among them, 824 metabolites were annotated after MS/MS analysis (~ 25% of total compounds). The annotation rate of human metabolomics analysis is approximately 10% [21, 22], suggesting that our annotation rate was reasonable and suitable for further investigations. The annotated compounds are listed in Supplementary file 1.
There were 667 compounds derived from positive and negative ion modes that exhibited significant changes between the IR and ID groups according to the volcano plots and VIP values (Fig. 2). Blue dots represent the upregulated compounds, whereas red dots indicate the downregulated compounds in IR cells compared to ID cells. The size of the dots indicates the VIP scores.
Our previous report suggested that glycolysis is highly active in ID T. vaginalis [23]. However, pyruvate would not be converted into acetyl-CoA for ATP and fatty acid production due to the global downregulation of genes/proteins inside hydrogenosomes under ID conditions [4, 5]. To better understand the fate of pyruvate upon iron depletion, we concentrated on pyruvate-related compounds in this study.
2. Saccharide metabolism
The first group of compounds that we focused on were oligosaccharides since they were altered significantly in ID cells. Figure 3 highlights the most accumulated oligosaccharides in ID cells compared to that of the IR control.
1.1 Accumulation of ɑ-1,4-linked oligosaccharides in ID cells
The left portion of Fig. 3 shows that isomaltose, maltotriose, and maltopentaose increased significantly, except maltose. These di-, tri-, and penta-saccharides were generated from either glucose polymerization or glycogen degradation. The expression of enzymes related to these reactions, isomaltase and ɑ-glucosidases, were increased in ID cells (Fig. 3). The expression of one glycogen phosphorylase (TVAG_348330) was slightly upregulated, whereas glycogen synthase was downregulated under iron-limited conditions (Fig. 3). According to the expression profiles of glycogen metabolic enzymes, we speculated that the direction of glycogen and oligosaccharide metabolism was toward degradation.
Glycogen is a storage saccharide reported to accumulate and be consumed rapidly in the log phase of axenic cultured T. vaginalis. The activity of glycogen phosphorylase exhibited the same trend as the glycogen level in the previous measurement [15]. Glycogen consumption along with more active glycolysis during the encystation process have been demonstrated in E. histolytica and G. lamblia [24–26]. In the case of iron shortage, the transition to pseudocysts and more active glycolysis implied that T. vaginalis underwent an encystation-like event similar to these intestinal protozoa. The possible utilization of glycogen not only for energy metabolism but also provides a substrate for cyst wall synthesis, such as chitin [25]. The true cyst wall is absent in the pseudocyst of T. vaginalis; however, it would be a catalytic direction from glucose to cellulose. This will be discussed in the following section.
1.2 Cellobiose accumulation in ID cells
Another disaccharide was highlighted because of the drastic elevation in ID cells, cellobiose, compared to the IR control (Fig. 3). Cellobiose is the building block of cellulose, a structural polymer for plants and cysts [12]. The enzyme responsible for the conversion between NDP-glucose and cellobiose, β-glucosidase, was downregulated in ID cells. Conversely, the upregulation of cellulase (TVAG_194310), which converts cellulose to cellobiose, was found in ID cells. Notably, cellulase catalyzes bidirectional reactions, which are also responsible for cellulose biosynthesis [27]. The evidence might imply that cellulase is involved in cellulose biosynthesis rather than breakdown to cellobiose.
The interconversion of cellobiose and polymeric cellulose is functionally associated with cell structure. Previous investigations demonstrated that polysaccharides, including chitin, acid-fast lipids, and cellulose, are the main backbone for cyst wall formation in distinct encysting protozoa [12]. Pseudocysts of T. vaginalis are built up with a chitin-/cellulose-based architecture [14]. Although there are 4 chitinase genes encoded by T. vaginalis, none of them have been evaluated. Based on our RNA expression dataset, all chitinases were not significantly altered between IR and ID conditions [5]. We first demonstrated the presence of cellobiose, a disaccharide with a β-1,4-glycosidic linkage of glucoses, which is also the substrate for cellulose biosynthesis. The accumulation of cellobiose was accompanied by downregulation of β-glycosidase and upregulation of cellulase in ID cells. Moreover, cellobiose is not used for the proliferation of trichomonad cells [16]. Collectively, we speculated that cellulose biosynthesis might be involved in the progression of ID cells during pseudocyst formation.
1.3 Raffinose family oligosaccharides (RFOs) accumulated in ID cells
We have stated above that storage glycogen and structural cellulose metabolism are affected by iron depletion. Surprisingly, our dataset showed that raffinose family oligosaccharides (RFOs), including sucrose, raffinose, and stachyose, also significantly accumulated under ID conditions (Fig. 3). RFOs are saccharides composed of fructose, glucose, and galactose. In the synthetic pathway, galactinol is first generated from UDP-galactose and myo-inositol by galactinol synthase. Galactinol and sucrose are substrates for raffinose synthesis catalyzed by ɑ-galactosidase. Stachyose is subsequently generated via the combination of raffinose and galactinol [28]. Levels of myo-inositol and galactinol were dramatically elevated in ID cells (Fig. 3, bottom right). The RNA levels of enzymes related to RFO synthesis, β-fructosidase (TVAG_254130) and ɑ-galactosidase (TVAG_145340), were also upregulated in ID cells. Although UDP-galactose was not detected and galactinol synthase was absent in T. vaginalis, evident elevations in galactinol and the downstream RFOs led us to hypothesize that trichomonad cells produced different forms of sugars to respond to unfavorable environments.
RFOs are broadly distributed in the kingdom of plants and are especially well studied in soybean and chickpea [18]. These oligosaccharides participate in several biological functions, such as carbon storage and transport, tolerance to environmental stresses, and human gut microbiome manipulation [18]. A previous investigation demonstrated that recombinant trichomonad β-fructosidase is capable of degrading sucrose and raffinose [17]. However, as a storage sugar, raffinose is not utilized for the growth of T. vaginalis [29]. These statements suggested that raffinose, sucrose, and stachyose were not likely to be used for energy production and cell proliferation.
RFOs accumulate under iron-depleted conditions, and they act as stress-responsive molecules in plants [30, 31]. The function of accumulated RFOs reflects the involvement of environmental changes in cases of changes in osmolarity, temperature, and infections [32]. Furthermore, RFOs possess hydroxyl radical scavenging activity that would be an important antioxidant [33]. We suggested that reactive nitrogen species (RNS) rather than reactive oxygen species (ROS) accumulated in ID trichomonad cells. The imbalance of redox homeostasis in iron-limited circumstances induce the thioredoxin-peroxiredoxin system in T. vaginalis [5]. Accordingly, we speculated that the increase in RFOs might also be an antioxidant induced by iron shortage.
Collectively, we hypothesized that glycogen was used as the resource for more active glycolysis; on the other hand, cellulose biosynthesis occurred at the same time for pseudocyst formation. RFO accumulation revealed a possible antioxidative or signaling role in response to iron shortage, which should be examined in detail in the future.
2 Lipid metabolism
Previous studies have demonstrated that T. vaginalis possesses defective machinery to synthesize lipid-related biomolecules, such as fatty acids [7]. The draft genome also showed that there are no complete fatty acid beta-oxidation or biosynthesis genes encoded by the protist [34]. However, we identified different kinds of lipids in the metabolomics analysis.
2.1 Reduction in C18 fatty acid levels in ID cells
Figure 4 shows that unsaturated fatty acid levels were mostly comparable, except for significant reductions in the contents of linoleic acid (C18), gamma-linolenic acid (C18), and erucic acid (C22) in ID cells. Additionally, a drastic decrease in the stearic acid (C18) content in ID cells was shown in the category of saturated fatty acids. These results indicated a greater utilization or lower synthesis of multiple C18 fatty acids in ID trichomonad cells.
Alterations in lipid metabolism might be involved in the morphological changes in human pathogenic protozoa, such as encystation. In G. lamblia, the composition of fatty acids has been investigated, and it was found that stearic (C18) and oleic (C18) acids are the most abundant fatty acids in Giardia during encystation [35]. T. vaginalis underwent a transformation from motile trophozoites to arrested pseudocysts under iron-limited conditions [13]. The composition of fatty acids in ID trichomonad cells (or pseudocysts) revealed a global decrease in C18 fatty acids (Fig. 4). The difference between previous and present studies is the stage of protists: encysting G. lamblia and pseudocysts of T. vaginalis. Moreover, lipid composition regulates the encystation process of E. histolytica [11, 36]. Hence, we hypothesized that these C18 fatty acids were likely incorporated into membranous structures during the progression of encystation.
Although transacylation activity participates in the generation of several lipids, previous studies revealed that T. vaginalis is unable to synthesize fatty acids [37]. However, medium-derived fatty acids (mainly medium-chain fatty acids (MCFAs)) are incorporated into phosphoglyceride and sphingolipids [7]. Indeed, the detectable phospholipids in our results were significantly reduced, especially derivatives of phosphoethanolamides (PEs) (Supplementary file 2). Derivatives of phosphatidylcholine, choline, and serine were not significantly changed in the metabolomics analysis (data not shown). Reductions in the detected fatty acids and phospholipids were likely incorporated into the membranous structure in T. vaginalis. Collectively, these results implied that iron shortage lowered most detected C18 fatty acids via putative fatty acid incorporation into phospholipids, which might be associated with pseudocyst formation in T. vaginalis.
2.2 Accumulation of capric acid in ID cells
Due to the lack of related enzymes in T. vaginalis, the composition of fatty acids had not been documented in detail previously. A gene annotated as 2-nitropropane dioxygenase precursor (TVAG_479680), also known as enoyl-acyl carrier protein reductase (ENR), participates in fatty acid biosynthesis [38]. ENR catalyzes the final step of fatty acid elongation of the type II fatty acid synthesis (FAS II). The expression of TvENR showed a significant elevation under ID conditions [5]. Here, we showed that a putative ENR-dependent synthesized capric acid was elevated under ID conditions, while most of them were comparable except stearic acid (discussed above) (Fig. 4B). This result suggested that a functional FAS II would exist in T. vaginalis, and the downstream fatty acid could be changed by iron depletion.
Acetyl-CoA is the essential building block of fatty acids, which was thought to be reduced in ID cells since the enzyme pyruvate:ferredoxin oxidoreductases (PFOs) decreased drastically in ID cells [5, 34]. We suggest that the accumulation of capric acid might utilize acetyl-CoA originating from neither glucose nor the remaining known substrates or generated from the degradation of longer fatty acids, such as stearic acid (C18) [37]. Nonetheless, it could still not be ruled out whether the transportation of fatty acids from the culture medium was induced by iron limitation.
Lipids are an important energy source, especially with limited sugar supplementation. Previous investigations revealed that enzymatic activities associated with β-oxidation are absent in T. vaginalis [37]. Here, we showed that at least three unsaturated fatty acids were significantly reduced in ID cells (Fig. 4). The mRNA expression of long-chain fatty acid acyl-CoA synthetase, the first enzymatic step in the oxidation of fatty acids, increased slightly, further implying that the usage of fatty acids by T. vaginalis [5]. However, this hypothesis should be verified in the future.
The only accumulated fatty acid identified in this study is capric acid (known as decanoic acid), an MCFA. MCFAs (composed of 7 to 12 carbons) have important biological functions in cells [39]. Previous reports demonstrated that MCFAs are crucial energy storage molecules. The relatively short carbon chain is an advantage for transportation throughout mitochondria and is therefore easier to catalyze [40]. Furthermore, less ROS are generated during MCFAs β-oxidation compared to long-chain fatty acids [41]. In addition to energy production, regulatory roles of MCFAs have been revealed, such as the enhancement of glycolysis in the brain [42]. This phenomenon reflects the fact that glycolysis is more active in T. vaginalis upon iron depletion [23]. Gluconeogenesis is also enhanced by MCFAs. However, the key enzymes are absent in T. vaginalis [34, 43]. Therefore, the accumulation of capric acid might be linked to the metabolic regulation of T. vaginalis under iron shortage.
Taken together, our results suggested that the utilization of C18 fatty acids was in response to the transformation of pseudocysts. Energy metabolism, such as glycolysis and fatty acid catalysis, was altered by iron depletion, which was possibly regulated by the significant accumulation of capric acid.
3 Amino acid metabolism
Catabolism of many amino acids relies on the presence of pyruvate. We demonstrated more active proteolysis via the ubiquitin‒proteasome system (UPS) triggered by iron shortage [5]. Moreover, several proteolytic enzymes were upregulated in ID cells. Proteolysis is responsible for the clearance and recycling of amino acids. We consequently assumed that amino acids would accumulate in trichomonad cells under iron-limited conditions. However, the dataset showed a decrease in amino acid levels in ID cells.
3.1 Alanine, glutamate, and serine were significantly reduced in ID cells
Among the detected amino acids, only alanine, glutamate, and serine were significantly reduced in ID cells (Fig. 5A). Alanine dehydrogenase and threonine dehydratase catalyze the conversion of alanine and serine to pyruvate, respectively. In addition, glutamate dehydrogenase is responsible for the conversion between glutamate and ɑ-ketoglutarate (Fig. 5B-D). It is noticeable that all these reactions were accompanied by the release of ammonia. Other amino acid catabolism associated with ammonia release, including arginine, asparagine, cysteine, glutamine, methionine, threonine, and tryptophan, were all reduced compared to the IR control, although without significance (Fig. 5B-D). Therefore, these amino acids were reduced with a similar upregulation trend as the corresponding enzymes in ID cells, implying that these reactions catalyzed in the opposite direction that generated ammonia.
Glutamate is the metabolic center for several amino acids [34]. Leucine and lysine showed unchanged or slightly elevated levels in ID cells. The enzyme catalyzes the conversion of glutamate to isoleucine, leucine, and valine, branched-chain amino acid (BCAA) aminotransferases (TVAG_026740), which are upregulated under ID conditions, while lysine aminotransferase is undetectable at the RNA level [5]. The slight increase in leucine and lysine implied that the catalytic directions of glutamate were not only toward ɑ-ketoglutarate but also toward leucine and lysine (Fig. 5).
Nitric oxide (NO) plays a crucial survival role in ID trichomonad cells; however, arginine-dependent NO generation occupied only ~ 20% of the total NO, indicating that most NO was synthesized via different unknown machineries [5]. Ammonia is a nitrogen-containing compound that can be converted to NO by oxidation [44, 45]. Ammonia metabolism takes place in the hydrogenosome of T. vaginalis by the activity of hydroxylamine reductase (HCP, TVAG_336320), which is highly expressed in ID cells [46]. Additionally, HCP is known to scavenge NO in the bacterial model, indicating a possible linkage between ammonia and NO metabolism in T. vaginalis [47]. Therefore, we hypothesized that ammonia derived from amino acid catabolism might be an important substrate for NO production in T. vaginalis under iron-depleted conditions.
3.2 Dipeptides significantly accumulated in ID cells
Most amino acids reduced in ID cells have been discussed above; however, whether mechanisms other than amino acid catabolism are involved is not clear. A fraction of dipeptides occupied the annotated compounds of the dataset and attracted our attention. Dipeptides are composed of two amino acids linked with peptide bonds and are generated either proteolytically or proteinogenically [48, 49]. There are 400 possible combinations of dipeptides theoretically. In the present investigation, we identified 170 dipeptides (Fig. 6). Among them, 33 accumulated in ID cells, whereas 9 were elevated in IR cells. This result could be an explanation, in part, for the overall amino acid level reduction in ID cells being caused by dipeptide accumulation. Dipeptides with N-terminal proline, valine, and phenylalanine were the most abundant in ID cells. In fact, the expressions of dipeptidases and dipeptidyl peptidases were mostly upregulated in ID cells; thus, the synthetic mechanism of dipeptides could not be determined [5].
Dipeptides composed of cysteine residues are linked to antioxidation activity [50]. As the imbalance of redox caused by iron depletion, however, the only identified cysteine-containing dipeptides, Ile-Cys and Lys-Cys, were not changed by the iron content in T. vaginalis. Dipeptides also play regulatory roles. The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is the target of the Tyr-Asp dipeptide. The interaction between GAPDH and Tyr-Asp results in the restriction of enzymatic activity [51]. This suggested that these stress-specific accumulated dipeptides might participate in biological regulation in T. vaginalis.
In summary, the overall reduction in the levels of amino acids in ID cells might be associated with the accumulation of dipeptides. In addition, significantly reduced alanine, glutamate, and serine were accompanied by the release of ammonia, which was likely the substrate for NO production in the protist under iron-deficient environments.