Introduction
Trehalose (α-d-glucopyranosyl α-d-glucopyranoside) is a non-reducing disaccharide where two glucose units are linked in an α,α-1,1-glycosidic linkage. Trehalose is present in a wide variety of organisms, including bacteria, yeast, fungi, insects, nematodes and shrimps, lower and higher plants. It does not occur in vertebrate cells (Elbein et al., Reference Elbein, Pan, Pastuszak and Carroll2003). In many organisms, it may serve as an energy source or as a signalling molecule to direct or control certain metabolic pathways, and even affects growth. In other organisms, it is induced by stress conditions, such as heat, drying and oxidative stress, and protects proteins and cellular membranes from inactivation or denaturation caused by these stresses (Behm, Reference Behm1997).
In the case of nematodes, trehalose serves a variety of functions, i.e. as a protectant for tissues and embryos during desiccation and freezing stress (Womerslay & Smith, Reference Womerslay and Smith1981; Crowe et al., Reference Crowe, Hoekstra and Crowe1992; Womerslay & Higa, Reference Womerslay and Higa1998; Wharton et al., Reference Wharton, Judge and Worland2000, Reference Wharton, Goodall and Marshall2002), as a low-molecular weight energy source (Castro & Fairbairn, Reference Castro and Fairbairn1969), to aid glucose uptake (Barrett, 1981; Behm, Reference Behm1997) and in the hatching mechanism of eggs (Perry, Reference Perry1989). This disaccharide could probably function as the principal circulating blood sugar in nematodes, similar to insects (Behm, Reference Behm1997; Thompson, Reference Thompson2003).
In most eukaryotes the metabolism of trehalose is catalysed by three enzymes: trehalose-6-phosphate synthase (TPS, EC2.4.1.15) and trehalose-6-phosphate phosphatase (TPP, EC3.1.3.12) are responsible for trehalose synthesis, while trehalase (TRE, EC3.2.1.28) catalyses the hydrolysis of the sugar (Pellerone et al., Reference Pellerone, Archer, Behm, Grant, Lacey and Somerville2003). A fourth enzyme, trehalose phosphorylase (TreP, EC2.4.1.64), is limited to a few microorganisms, fungi and Euglena gracilis, where it catalyses a reaction that breaks down trehalose in the presence of inorganic phosphate, transfers one glucose to inorganic phosphate in order to produce glucose-1-phosphate and releases the other glucose as a free sugar (Maréchal & Belecopitow, Reference Maréchal and Belecopitow1972; Elbein et al., Reference Elbein, Pan, Pastuszak and Carroll2003). However, in Agaricus bisporus, TreP catalyses the reversible reaction, of both degradation (phosphorolysis) and synthesis of trehalose (Wannet et al., Reference Wannet, Camp, Wisselink, Drift, Griensven and Vogels1998). In Ascaris suum, both disaccharide catabolism enzymes – TRE and TreP – participate in trehalose breakdown (Dmitryjuk & Żółtowska, Reference Dmitryjuk and Żółtowska2004).
Trehalose biosynthesis pathways are widely distributed in nature, except in vertebrates. There are five known trehalose biosynthetic routes: TPS/TPP, TS, TreY/TreZ, TreP and TreT pathways. In invertebrates, only the first pathway (TPS/TPP) is known to exist (Avonce et al., Reference Avonce, Mendoza-Vargas, Morett and Iturriaga2006).
The synthesis of trehalose in nematodes proceeds in the classical pathway and is catalysed by the action of two enzymes: TPS, which catalyses the transfer of glucose from uridine diphosphate (UDP)-glucose to glucose-6-phosphate to produce trehalose-6-phosphate (T6P); and TPP, which can convert T6P to free trehalose and Pi (Behm, Reference Behm1997). The properties of TPS were studied in Aphelenchus avenae (Loomis et al., Reference Loomis, O'Dell and Crowe1980). Trehalose metabolism genes have also been studied in Caenorhabditis elegans, filarial nematodes (Pellerone et al., Reference Pellerone, Archer, Behm, Grant, Lacey and Somerville2003) and in A. avenae (Goyal et al., Reference Goyal, Browne, Burnell and Tunnacliffe2005).
The distribution and metabolism of glycogen, the main energy source in nematodes, has been well studied by a number of authors (Dubinský et al., Reference Dubinský, Ryboš and Turčeková1980; Turčeková et al., Reference Turčeková, Zemek, Dubinský and Ryboš1985, Reference Turčeková, Zemek, Dubinský and Ryboš1986; Żółtowska, Reference Żółtowska2001). Trehalose metabolism is closely linked with glycogen metabolism, as two intermediates, G6P and UDPG, are common to both (Behm, Reference Behm1997). In the present study, we have focused on the synthesis of trehalose. This is a revision of studies done by Fairbairn & Passey (Reference Fairbairn and Passey1957) and Feist et al. (Reference Feist, Read and Fisher1965) about the distribution and synthesis of trehalose in Ascaris. We have tried to define the distribution patterns of both trehalose anabolism enzymes and the product of synthesis (trehalose) in A. suum tissues. In addition, we looked at levels of glucose, that is, the component of trehalose. Trehalose may be acting as a regulatory molecule in the control of glucose metabolism in the studied parasite.
Materials and methods
The material for the study consisted of adult female A. suum. The extracts were prepared from cuticle, muscles, intestines and parts of the reproductive system – ovary, oviduct and upper, central and lower length of uterus. Haemolymph was also collected for the tests. The tissues, besides haemolymph, were homogenized with 0.9% NaCl at 1:4 w/v in a YellowLine DI 18 basic homogenizer (Ikawerke, Germany). The homogenates and haemolymph were centrifuged at 1500 × g for 10 min. Trehalose and glucose, activity of TPS and TPP, as well as protein contents were identified in the supernatant.
Activity of TPS was identified according to Giaever et al. (Reference Giaever, Stryvold, Kaasen and Strøm1988) and TPP activity was determined using the method of Kaasen et al. (Reference Kaasen, Falcenberg, Stryvold and Strøm1992). For TPS from muscles a buffer at pH 4.2 was used. For other tissues, buffer at pH 7.0 was employed. The end product of reactions and content of sugars in tissues were determined by using high-performance liquid chromatography (HPLC). To prepare the samples for HPLC analysis, samples were first boiled for 5 min, two volumes of ethanol were added and centrifuged. Supernatants were desiccated at 50°C and dissolved in acetonitrile/deionized water (3:2, v/v) mixtures and filtered using nylon Micro-Spin Filter Tubes (Alltech Associates, USA). For each sample, 20 μl was injected into a Shimadzu SCL-10A system using refractive detector RID 10A (Kyoto, Japan). The high-performance carbohydrate cartridge column (4.6 × 250 mm; Waters, The Netherlands) was eluted with acetonitrile:degassed and deionized water (75/25%, 1 ml/min) and kept at 35°C during analysis. The concentrations of trehalose and glucose were analysed using Chromax 2005 software (POL – LAB; Warsaw, Poland).
The activity of both enzymes was expressed in units (U) per mg of proteins marked by Bradford's method (Bradford, Reference Bradford1976). The enzymatic unit, U, represents the quantity of enzyme that produces 1 nmole trehalose at 37°C, in 1 min. The content of trehalose and glucose was expressed in mg per 1 g of wet tissue. The results are an average of nine replicates. Statistical analysis was done using Statistica 8 program (StatSoft Inc., Tulsa, Oklahoma, USA).
Results
The level of trehalose and glucose in tissues of Ascaris suum
Trehalose occurs in the muscles, reproductive system and haemolymph of the parasite. The lowest concentration of trehalose was found in muscles. A higher amount of the disaccharide was detected in the reproductive system as a whole, and almost tenfold higher in haemolymph compared to muscles. Trehalose does not accumulate in the cuticle and intestine of the nematode (table 1). The level of glucose was the highest in haemolymph. In comparison to trehalose, glucose was detected in a huge concentration in the cuticle and intestine. A lower amount of glucose was found in the reproductive system as a whole (table 1).
Table 1 The level of trehalose and glucose in individual tissues of Ascaris suum.
a Mean ± SD; n = 9; **P < 0.01; *P < 0.05.
The level of trehalose and glucose in parts of the reproductive system of the nematode
A low level of both sugars (trehalose and glucose) was found in the ovary. Compared to the ovary, a slightly lower level of trehalose and twofold higher level of glucose was seen in the oviduct. The level of trehalose increased fourfold in the upper uterus, and the level of glucose decreased below levels in the ovary. The highest level of trehalose and glucose was observed in the middle uterus. The levels of both sugars decreased unexpectedly in the lower uterus, still the level of trehalose was twofold higher than in the upper uterus (table 1).
The activity of trehalose synthesis enzymes in tissues of Ascaris suum
Both trehalose synthesis enzymes (TPP and TPS) were found to be active in muscles, haemolymph and reproductive system. Synthesis of trehalose was not observed in cuticle and intestine of the parasite (fig. 1). The highest activity of TPS was detected in muscles (300.39 ± 107.33 U/mg). A slightly lower activity was found in the reproductive system (289.83 ± 253.37 U/mg). The activity in haemolymph was one-third lower (206.45 ± 117.4 U/mg) than in the muscles. Similar results were found for TPP. The highest activity of trehalose-6-phosphate phosphatase was detected in muscles of the nematode (646.078 ± 222.819 U/mg). TPP from the reproductive system had slightly lower activity (462.97 ± 376.33 U/mg) and the lowest activity of TPP was in the haemolymph (200.87 ± 92.63 U/mg). The activity of TPP was much higher than TPS in muscles and reproductive system. Activity of both enzymes was almost on the same level in the haemolymph (fig. 1).
Fig. 1 The activity of trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) in tissues of Ascaris suum, ■ TPS; □ TPP.
The activity of trehalose synthesis enzymes in the reproductive system of the nematode
The activity of both enzymes (TPS and TPP) was found to be decreased in oviduct (TPS: 34.38 ± 7.61 U/mg; TPP: 105.9 ± 39.03 U/mg) compared with the ovary. The activity increased in the upper (TPS: 258.45 ± 80.93 U/mg; TPP: 314.81 ± 77.31 U/mg) and middle part of the uterus (TPS: 274.39 ± 99.09 U/mg; TPP: 350.65 ± 56.63 U/mg). Activity was especially high in uterus containing fertilized eggs (TPS: 803.5 ± 88.64 U/mg; TPP: 866.29 ± 511.66 U/mg) (fig. 2).
Fig. 2 The activity of trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) in individual parts of the female reproductive system of Ascaris suum, ■ TPS; □ TPP.
The activity of TPP was higher than TPS in all studied parts of the reproductive system. Huge differences were observed in the ovary: TPP (357.4 1 ± 123.25 U/mg) and TPS (39.92 ± 13.41 U/mg) (fig. 2).
Discussion
Trehalose is accumulated in many representatives of all systematic (taxonomic) groups, except vertebrates. Fairbairn & Passey (Reference Fairbairn and Passey1957) studied distribution and occurrence of trehalose in eggs and adult Ascaris, but the mechanism of trehalose biosynthesis is not fully understood in nematodes. In insects, trehalose is present in high concentrations as the main haemolymph (blood) sugar. Fructose 2,6-bisphosphate is a key metabolic signal in the regulation of trehalose synthesis in insects. Synthesis of trehalose in the fat body of insects is stimulated by hypertrehalosaemic hormones, which cause a decrease in the content of fructose 2,6-bisphosphate in fat body cells. It is a potent activator of the glycolytic enzyme 6-phosphofructokinase-1 and a strong inhibitor of the gluconeogenic enzyme fructose 1,6-bisphosphatase (Becker et al., Reference Becker, Schloder, Steele and Wegener1996; Thompson, Reference Thompson2003).
Trehalose and its intermediate T6P can be very important regulators of sugar metabolism. In higher plants, trehalose may be potentially toxic or inhibitory, when correlating with high activity of trehalase (Veluthambi et al., Reference Veluthambi, Mahadevan and Maheshwari1981), but certain other plants are considered to synthesize trehalose. In Arabidopsis, trehalose is present and T6P levels are likely to be tightly controlled by the relative activities of TPS, TPP and trehalase (Wingler, Reference Wingler2002; Eastmond et al., Reference Eastmond, Li and Graham2003). In plants, T6P is emerging as a significant sugar signal that regulates sugar utilization and starch metabolism and interacts with other signalling pathways (Lunn et al., Reference Lunn, Feil, Hendriks, Gibon, Morcuende, Osuna, Scheible, Carillo, Hajirezael and Stitt2006; Paul, Reference Paul2007). Research in other organisms has shown that too much T6P can be toxic, for example in the pathogenic fungus Cryptococcus neoformans (Petzold et al., Reference Petzold, Himmelreich, Mylonakis, Rude, Toffaletti, Cox, Miller and Perfect2006).
The TPS/TPP pathway exists in nematodes, involving the intermediate T6P. The regulation of trehalose metabolism can be also connected with this intermediate. In C. elegans, two TPS and five putative trehalase genes were identified by searching the genome sequence. Several putative homologous genes in the filarial nematodes Brugia malayi and Onchocerca volvulus have also been identified, which suggests important functions for these genes in nematode physiology, as trehalose is claimed to be important, functioning in sugar transport, energy storage and protection against environmental stresses (Pellerone et al., 2003) T6P is also important and may be potentially lethal for the metabolism of trehalose in C. elegans. Kormish & McGhee (Reference Kormish and McGhee2005) identified one particular gene, gob-1, that, when mutated, produces the gut-obstructed Gob phenotype. It is interesting that gob-1 encodes the C. elegans enzyme TPP. The importance of trehalose anabolism and its regulation mechanism in other nematodes, such as A. suum is unknown, whereas the catabolism of trehalose in this nematode has already been researched (Dmitryjuk & Żółtowska, Reference Dmitryjuk and Żółtowska2003, Reference Dmitryjuk and Żółtowska2004; Dmitryjuk et al., Reference Dmitryjuk, Żółtowska and Łopieńska-Biernat2005). Until now, activity of TPS and TPP has not been studied in this parasite. The issue of synthesis of trehalose in tissues of Ascaris was studied by Feist et al. (Reference Feist, Read and Fisher1965), but the activity of both trehalose synthesis enzymes was not demonstrated. Our studies are a revision and update of earlier data with the application of modern techniques.
In our experiment, we have shown the occurrence of trehalose in muscles, haemolymph and in all studied parts of the female reproductive system. Trehalose was not detected in cuticle and intestine of the parasite. Glucose was observed in all studied tissues. No occurrence of trehalose in the intestine of Ascaris was also reported by Fairbairn & Passey (Reference Fairbairn and Passey1957). However, they detected trehalose in the remaining tissues (e.g. in integument).
Our data of distribution of trehalose in the body of the nematode was correlated with the occurrence of active enzymes were involved in the synthesis of trehalose. The TPS and TPP enzymes were active in all the studied tissues, except cuticle and intestine. The study done by Feist et al. (Reference Feist, Read and Fisher1965) showed a high possibility of trehalose synthesis in the reproductive tissues and a very low possibility in the muscles of adult nematodes. We did not observe the synthesis of trehalose in intestine and in half of the haemolymph samples. In our study, the activity of TPS and TPP was the lowest in haemolymph. Probably, the lack of synthesis of trehalose in studies done by Feist et al. (Reference Feist, Read and Fisher1965) was the result of dilution of haemolymph in a 1:1 ratio.
Despite the low enzyme activity of trehalose synthesis in haemolymph, we observed the highest concentration of trehalose here, amongst all studied tissues. The level of glucose in haemolymph was 4.5-fold lower than that of trehalose. In earlier research we did not observed the activity of trehalase in haemolymph of Ascaris (Dmitryjuk & Żółtowska, Reference Dmitryjuk and Żółtowska2004). According to Behm (Reference Behm1997), the high level of trehalose and low concentration of free glucose, combined with T6P synthase and phosphatase activity and the lack of trehalase activity, indicates that trehalose functions as a circulating sugar in Ascaris. Above-mentioned observations support the possiblity that the disaccharide may be acting as a regulatory molecule in the control of glucose metabolism in the researched parasite. In insects, a similar correlation was observed, where trehalose is the main haemolymph sugar. There are also high concentrations in thorax muscles, where the disaccharide provides energy required for flying, by the participation of trehalase (Wyatt & Kalf, Reference Wyatt and Kalf1957; Becker et al., Reference Becker, Schloder, Steele and Wegener1996).
These observations do not confirm the results obtained by Fairbairn & Passey (1957), who detected the highest concentration of trehalose in female muscles and almost a 2.5-fold lower concentration in the haemolymph of Ascaris.
In this study, synthesis of trehalose was not observed in the intestine and cuticle of the parasite. However, a relatively high level of glucose was observed in these tissues. The high level of glucose in both tissues was also observed by Fairbairn & Passey (1957). This phenomenon could be due to high activity of enzymes that catabolize trehalose, TRE and TreP (Dmitryjuk & Żółtowska, Reference Dmitryjuk and Żółtowska2004). Loss of TPS activity can result in an inability to metabolize hexoses, especially glucose. This phenomenon was observed in Saccharomyces cerevisiae (Gancedo & Flores, 2004). Lack of accumulation of trehalose and activity of trehalose breakdown enzymes in intestine and cuticle (Dmitryjuk & Żółtowska, Reference Dmitryjuk and Żółtowska2004) could enable disaccharides to pass through biological membranes.
An almost sevenfold lower activity in the synthesis of trehalose in muscles than in ovary–oviduct was observed using buffer with pH 7.4 (Feist et al., Reference Feist, Read and Fisher1965). In our studies, the residual activity for TPS from muscles was found using a buffer with neutral pH. A tenfold increase of enzymatic activity was observed after the change to acidic pH (pH 4.2). An acidic pH of reaction seems to be appropriate for the achievement of maximal enzymatic activity, when the conditions prevailing in vivo in muscles of the parasite are taken into consideration. Trehalase extracted from muscles of Ascaris also worked at acidic pH (pH 4.9), (Dmitryjuk & Żółtowska, Reference Dmitryjuk and Żółtowska2003).
Probably, trehalose has a special function in developing oocytes in the reproductive system. In the present studies, the level of trehalose in ovary was relatively low and was correlated with the low activity of TPS. The level of glucose was also very low. Trehalose levels decreased slightly in the oviduct. This decrease was correlated with the decrease of TPS and TPP activities. However, the level of glucose was almost twofold higher than in the ovary. Probably, the activity of trehalase that comes from the higher demand for glucose in contractile muscle fibres of oviduct enables the movement of eggs.
Activity of TPS and TPP gradually increased in the fragments of uterus. In addition, the level of trehalose was the highest here. The activity of TPS and TPP reached the highest level amongst studied tissues in the lower part of the uterus. We suggest that trehalose is an important carbohydrate for mature oocytes, which need trehalose in the external environment. This hypotheses is confirmed by other studies (Dmitryjuk et al., Reference Dmitryjuk, Żółtowska, Kubiak and Głowińska2006) that indicate a high concentration of trehalose (2.96 ± 0.07 mg/g) in zygotes, during all periods of embryogenesis and in L2 larvae. Accumulated trehalose in eggs is needed during hatching (Perry, Reference Perry1989). According to Fairbairn & Passey (1957), trehalose is accumulated in perivitelline fluid, whereas glycogen is required in developing embryos.
Interestingly, the activity of trehalose synthesis enzymes was the highest in the lower uterus, but the level of disaccharide decreased almost twofold in the middle uterus. However, we observed the highest level of glucose in the middle uterus amongst the other studied parts of the reproductive system of the female nematode. Moreover, based on the high share of eggs to walls of the uterus, we can conclude that trehalose is most intensively used in mature oocytes. This hypothesis seems to confirm a high activity of trehalase in eggs, which were extracted from the end fragments of the nematode uterus (Dmitryjuk et al., Reference Dmitryjuk, Żółtowska, Kubiak and Głowińska2006).
Conclusions and perspectives
We suggest that besides glycogen, trehalose is an important carbohydrate for growth and development of A. suum. It occurs in a high amount in haemolymph, muscles, and the reproductive system of the parasite and certainly is not toxic. In these tissues, we also observed active enzymes (TPS and TPP), which take part in the synthesis of trehalose. Trehalose and its synthesizing enzymes do not occur in intestine and cuticle, whereas we could detect relatively high levels of glucose in these tissues. Since the level of disaccharide decreases from the distal uterus to the level of the middle uterus, we suggest that trehalose is highly used by mature oocytes, but still plays a role of energy source for eggs after their passage to the external environment. In general, we can deduce that trehalose, besides glycogen, is the most important storage sugar and protectant for Ascaris, facilitating the life cycle of the parasite.
In the future it will be very important to get to know other aspects of regulation of trehalose metabolism in Ascaris. Currently, we are working on the biochemical and genetic characterization of A. suum muscle TPS. We hope that this will answer a number of questions related to the physiology of Ascaris, especially those related to the regulation of sugar metabolism in the parasite, and the meaning of a disaccharide that, contrary to host metabolism, plays a key role in nematode physiology.
Acknowledgements
This research was supported by the Ministry of Education and Science, grant no. 2PO4C 124 29, from 2005 to 2008.
