Metabolites in the snail

One of the first nuclear magnetic resonance (NMR) spectroscopy studies on the tissues of B. glabrata by 31P nuclear magnetic resonance spectroscopy revealed that several phosphorus metabolites were affected by S. mansoni infection. The decrease in a phosphonic acid was particularly notable. Although the ATP/ADP ratio was not affected by infection, an increase in the relative level of inorganic phosphate suggested a possible decrease in phosphorylation potential once the snail has been infected (Thompson and Lee, Reference Thompson and Lee1985).

A validation study was performed by Fried et al. (Reference Fried, Muller, Broadway and Sherma2001) to study B. glabrata when infected with S. mansoni and maintained on a diet of hen's egg yolk or Romaine lettuce. A number of neutral lipids were analysed in the digestive gland-gonad complex (DGG) of snails maintained on both diets. Infected snails maintained at 26 C and fed either diet produced fully developed cercariae by 4 wk post-infection (PI). Likewise, infected snails maintained at 23 C and fed either diet produced fully developed cercariae by 6 wk PI. Correlation between diet and time to cercarial patency was not proven. However, the analysis of neutral lipids showed that the DGG of infected snails fed the yolk diet contained greater amounts of free sterols and cholesteryl esters but not triacylglycerols compared to infected snails fed the lettuce diet (Fried et al. Reference Fried, Muller, Broadway and Sherma2001).

Also the effects of aestivation or starvation on the neutral lipid and phospholipid content of B. glabrata infected with S. mansoni were studied six years later. Infected-aestivated snails exhibited greater mortality rate and weight loss after 7 days than did the infected-starved snails. The steryl ester concentration in the infected-starved snails was increased compared with the controls but not compared with infected-aestivated snails; the concentration of phosphatidylcholine in infected-aestivated snails was significantly decreased compared with the controls but not when compared with the infected-starved snails. It was concluded that aestivation or starvation had a significant effect on the concentration of certain lipid classes in the DGG of B. glabrata infected with S. mansoni (White et al. Reference White, Fried and Sherma2007). Similarly a set of carboxylic acids were quantified in the DGG and haemolymph of B. glabrata. Infection with S. mansoni caused a significant reduction (Student's t test, P<0·05) in the concentrations of acetic, fumaric, malic and pyruvic acids in the DGG but not the haemolymph of B. glabrata compared to uninfected cohort snails. The findings suggested that these acids are utilized by the sporocysts and cercariae in the snail tissue or that the infection stimulates reduced production or increased utilization by the snail tissue (Massa et al. Reference Massa, Chejlava, Fried and Sherma2007).

Metabolites in the cercaria-worm-host interaction cycle

Haas et al. (Reference Haas, Haeberlein, Behring and Zoppelli2008) have found that cercariae of S. mansoni approach the host's skin via chemokinesis which can be used to calculate the attraction of cercarie to certain metabolites. Cercarie respond to human skin extracts by swimming in shifts which the group quantified as the rate of change of direction (RCD). Molecules termed attractants were identified by fractionating human skin surface extracts. The cercariae responded specifically to three skin surface compounds by increasing the frequency of shifts in swimming direction. (1) Free fatty acids. The effectiveness was limited to chain lengths between 10 and 14 carbon atoms in saturated chains and increased by the number of double bonds. (2) Free L-arginine at concentrations as low as 100 nM. (3) Small peptides with terminally located L-arginine at concentrations as low as 50 μM (Haas et al. Reference Haas, Haeberlein, Behring and Zoppelli2008; Haeberlein and Haas, Reference Haeberlein and Haas2008).

Although not specifically a metabolite, but due to the rarity of cercarie studies also mentioned in this review, a small protein (8 kDa) was recently determined by matrix-assisted laser desorption/ionization time-of-flight (ToF). This technique has been widely applied to the identification of proteins related to infection and in this particular case the small calcium-binding protein (SjCa8) was specifically expressed in cercariae and skin-stage schistosomulum (transformed within 3 h) (Lv et al. Reference Lv, Yang, Hu, Sun, He, He, Li, Zhou, Fung, Yu, Zheng, Cao and Wu2009). Another small protein (55 kDa) was detected in adult worm extracts and was also found to be expressed on the tegumental surface of cercariae (Lochmatter et al. Reference Lochmatter, Schifferli and Martin2009).

One of the first studies published concerning detection of various metabolites concentrated on the uptake of sugars and amino acids by the adult worm. Transintegumental uptake in living schistosomes (in vitro) was tested by using ¹⁴C labeled compounds. L-glucose uptake was low, but high uptake rates were observed in both sexes for D-glucose. Minimal uptake of mannitol was observed. The uptake rates of D-glucose and the L-amino acids (arginine, ornithine, lysine, histidine, phenylalanine and serine) were higher in female schistosomes. Slight elevations in uptake by females were observed for threonine, valine and glycine, but aspartate uptake was slightly higher in males. No dramatic male-female differences were immediately apparent for the uptake of proline, leucine, isoleucine, tyrosine and glutamate. Schistosome uptake of L-amino acids that are essential for vertebrates was generally higher than uptake of the non-essential amino acids (Cornford and Oldendorf, Reference Cornford and Oldendorf1979).

In an interesting study by Zhu et al. (Reference Zhu, Baggerman, Secor, Casares, Pryor, Fricchione, Ruiz-Tiben, Eberhard, Bimi and Stefano2002) HPLC coupled with electrochemical detection and by nano-electrospray-ionization-TOF analysis were employed to show that adult Dracunculus medinensis and S. mansoni both contain the opiate alkaloid morphine and that D. medinesis also contained the conjugated metabolite, morphine 6-glucuronide. From these results, it was suggested that helminths could use opiate alkaloids as immunosuppressive and anti-nociceptive signal molecules, to down-regulate immunosurveillance, responsiveness and pain signalling in their hosts (Zhu et al. Reference Zhu, Baggerman, Secor, Casares, Pryor, Fricchione, Ruiz-Tiben, Eberhard, Bimi and Stefano2002).

More recently, in a study concerning enzymes, cryosections of adult worms were probed and the production of calcium-mobilizing metabolites such as cyclic adenosine diphosphate ribose (cADPR) was detected. This molecule was synthesized by a novel member of the ADP-ribosyl cyclase family also expressed in S. mansoni (Goodrich et al. Reference Goodrich, Muller-Steffner, Osman, Moutin, Kusser, Roberts, Woodland, Randall, Kellenberger, LoVerde, Schuber and Lund2005).

Metabolites in liver

In the 1940s and 1950s, the first findings on the effects of S. mansoni infections on certain liver functions associated with protein metabolism were made. The metabolite allantoin increased in the rabbit on infection (Nishizaki et al. 1938) and fifteen years later, it was postulated in the mouse model that the urea cycle and its metabolism were disrupted (Daugherty et al. Reference Daugherty, Garson and Heyneman1954). In the 1980s, macrophages isolated from liver granulomas in mice infected with S. mansoni for 8 to 20 weeks, were found to synthesize thromboxane A2. Thromboxane A2 was the predominant metabolite found when the cells were stimulated by phagocytic stimuli, such as zymosan or the exogenous substrates arachidonic acid and PGH2 (Tripp et al. Reference Tripp, Needleman, Kassab and Weinstock1988).

More recently, Muller et al. (Reference Muller, Rosa Brunet, Fried and Sherma2001) studied lipid metabolism disruption due to infection, as it was suggested by the group that granulomatous lesions interfere with normal organ functions. Egg-induced tissue damage in a mouse model was investigated and the lipid profiles of the liver and ileum were analysed. The group found mainly that triacylglycerol and cholesteryl ester levels decreased significantly as the infection progressed (Muller et al. Reference Muller, Rosa Brunet, Fried and Sherma2001). Five years later, an original study claimed that praziquantel therapy improves granulomas found in the liver. The authors postulate that adult schistosomes excrete pigments after erythrocyte ingestion; these unidentified pigments are essentially small molecules and metabolites that are deposited outside and inside the granuloma. The group found correlation between the level of pigment and the degree of liver fibrosis. After dosing with praziquantel, results showed that the amount of pigment decreased and the size of granuloma diminished also. They concluded that schistosome infection and the effect of praziquantel can be monitored by the amount and distribution of the pigments (Tao et al. Reference Tao, Cai, Zhang, Huang, Xiang and Liu2006).

In recent years it has been suggested that chronic exposure to S. mansoni could have beneficial effects such as preventing the onset of diseases such as diabetes mellitus, but this validation study found different results. With four groups of mice: (1) control group, (2) group infected with S. mansoni, (3) group injected with streptozotocin to induce diabetes, and (4) group infected and then 3 months post-infection injected with streptozotocin. No differences were detected between the infected non-diabetic and infected diabetic groups regarding worm burden, tissue egg count and oogram. Consecutively, the group showed that S. mansoni infection reduced the rate of glucose uptake by the diaphragm (Thabet et al. Reference Thabet, Saleh, Thabet, Abdel-Aziz and Kalleny2008).

Based on the beneficial influence of the naturally occurring hormone, melatonin, on the immune system its administration was studied during the course of schistosomiasis. Melatonin was given with S. mansoni adult worm antigen (SWAP) and cercarial antigen (CAP). Results showed that the worm reduction percentages were 99·3% for SWAP+melatonin in the hamster model. Highly significant reductions in egg load in the liver were observed in the groups that received melatonin treatment suggesting a possible role for melatonin in egg production metabolism (Soliman et al. Reference Soliman, El Shenawy and El Arabi2008).

Recently, in a comparison of two different strains of mice, a significant increase in malondialdehyde level was found in infected mice. This reactive metabolite occurs naturally and is a marker for oxidative stress. Detailed discussion on the parasitological and other biochemical differences between the two strains was also presented in this manuscript (Bin Dajem et al. Reference Bin Dajem, Mostafa and El-Said2008).

Metabolites from eggs in human urine

The group of Hokke and Deelder have produced pioneering work with human urine. Free fuco-oligosaccharides (higher mass than a metabolite again, 1000–2000 uma but considered small molecule markers by the authors) were detected with matrix-assisted laser-desorption-ionization time-of-flight (MALDI-TOF). Urine samples from Egyptian individuals with different intensities of infection were analysed and correlated to schistosome egg load (Robijn et al. Reference Robijn, Koeleman, Hokke and Deelder2007, Reference Robijn, Planken, Kornelis, Hokke and Deelder2008). One can observe in Fig. 1 the results of analysis by MALDI-TOF: in particular the spectra of mAb 114-4D12-affinity-purified marker oligosaccharides from a pool of urine from S mansoni-infected individuals (A–C) and non-infected controls (D) are shown.

Fig. 1.

Matrix-assisted laser-desorption-ionization time-of-flight (MALDI-TOF) spectra of mAb 114-4D12-affinity-purified marker oligosaccharides from a pool of urine from heavily Schistosoma mansoni-infected individuals (A–C) and non-infected controls (D). The compositions of the marker oligosaccharides are indicated (A). H, hexose; N, N-acetylhexosamine; F, fucose. For quantification, the eluate of the S. mansoni-infected urine was spiked with 125 fmol (B) and 62·5 fmol (C) of the di-antennary N-glycan standard, H₅N₄ (m/z 1663·6, [M+Na]⁺). (Permission granted)

Metabonomics

NMR spectroscopy, coupled with data-mining techniques, is a powerful technique for metabonomics. This approach when compared with LC-MS-based studies shows greater reproducibility/robustness and samples can be reanalyzed. For metabolite identification some open-source software is available (Wang et al. Reference Wang, Shao, Chu, Ren, Mu, Qu, He, Jin and Xia2009). As mentioned previously, in Reference Wang, Holmes, Nicholson, Cloarec, Chollet, Tanner, Singer and Utzinger2004 Wang et al. pioneered the first application of metabonomics in parasitology. The study consisted of designing an experiment for the S. mansoni-mouse model which was analysed with ¹H NMR spectroscopy and multivariate pattern recognition. Mice were infected with 80 S. mansoni cercariae each and urine was collected for 49 and 56 days post-infection. They found that the metabolic signature of an S. mansoni infection consists of reduced levels of the tricarboxylic acid cycle intermediates, including citrate, succinate and 2-oxoglutarate, and increased levels of pyruvate, suggesting stimulated glycolysis. In addition a disturbance of amino acid metabolism was also associated with an S. mansoni infection, as indicated by depletion of taurine, 2-oxoisocaproate and 2-oxoisovalerate and elevation of tryptophan in the urine. Microbial-related metabolites were also detected: trimethylamine, phenylacetylglycine, acetate, p-cresol glucuronide, butyrate, propionate, and hippurate, were correlated with the S. mansoni infection, indicating disturbances in the gut microbiota (Wang et al. Reference Wang, Holmes, Nicholson, Cloarec, Chollet, Tanner, Singer and Utzinger2004).

The same team performed a similar metabolic profiling study to investigate the metabolic responses of Syrian hamsters to S. japonicum infection using ¹H NMR and pattern recognition. In two independent experiments, male hamsters were each infected with 100 S. japonicum cercariae. At days 34 and 36 post-infection, urine was obtained from hamsters and the respective controls housed individually in metabolism cages. The main biochemical effects of S. japonicum infection in the hamster consisted of reduced levels of urinary tricarboxylic acid cycle intermediates, including citrate and succinate and increased levels of pyruvate. In addition, as in the previous study a range of microbial-related metabolites, such as hippurate, p-cresol glucuronide, phenylacetylglycine and trimethylamine were also associated with S. japonicum infection. The major distinguishing result of S. japonicum infection in the hamster was the inhibition of manufacture or utilization of short-chain fatty acids, when compared to a S. mansoni infection in the mouse (Wang et al. Reference Wang, Utzinger, Xiao, Xue, Nicholson, Tanner, Singer and Holmes2006). Further to this, the group examined different tissue samples recovered from the host animal (mice) using the same metabolic profiling strategy. At day 74 PI, jejunum, ileum, colon, liver, spleen and kidney samples were collected (Li et al. Reference Li, Holmes, Saric, Keiser, Dirnhofer, Utzinger and Wang2009). Most tissues obtained from S. mansoni-infected mice were characterized by high levels of amino acids, such as leucine, isoleucine, lysine, glutamine and asparagine. High levels of membrane phospholipids metabolites, including glycerophosphoryl choline and phosphoryl choline were found in the ileum, colon, liver and spleen of infected mice.

S. mansoni has been fingerprinted using another technique, capillary electrophoresis (CE). CE is a technique that is mainly used for larger molecules but has seen an increased number of applications in metabonomics in the last years due to its low cost (Garcia-Perez et al. Reference Garcia-Perez, Vallejo, Garcia, Legido-Quigley and Barbas2008 a; Papaspyridonos et al. Reference Papaspyridonos, Garcia-Perez, Angulo, Domann, Vilca-Melendez, Heaton, Murphy, Holmes, Barbas and Legido-Quigley2008). Methods developed for CE-UV detection usually rely on charged compounds and are based on the mass-to-charge ratio of the metabolite in solution. In such a way cations and anions are detected. Application of a voltage across the capillary causes electrophoretic and electrosmotic movement, this results in the ionic species in the samples moving along the capillary and passing through the online UV detector. The Institute for Advanced Biosciences, Japan has led the way to the linking of CE-MS for metabolic fingerprint acquisition; its application to the field of parasitology could indeed bring new information on metabolites related with infection, since the group claims to have a database of 1000 metabolites detected already in bacteria (Soga and Imaizumi, Reference Soga and Imaizumi2001; Sugimoto et al. Reference Sugimoto, Kikuchi, Arita, Soga, Nishioka and Tomita2005; Monton and Soga, Reference Monton and Soga2007; Soga, Reference Soga2007; Soga et al. Reference Soga, Igarashi, Ito, Mizobuchi, Zimmermann and Tomita2009).

In CE, combining two analyses can enhance fingerprints when using only CE-UV. Two modes of separation were used with untreated urine samples. Micellar EKC (MEKC) and zonal CZE conditions where neutral compounds are also separated due to interaction with micelles. The traditional problems associated with variability in electrophoretic peak migration times for analytes were countered by using a dynamic programming algorithm for electropherogram alignment. Discriminant analysis (PLS-DA) provided a valuable means of rapid sample classification when infected urines were compared to controls (Garcia-Perez et al. Reference Garcia-Perez, Whitfield, Bartlett, Angulo, Legido-Quigley, Hanna-Brown and Barbas2008b). In Fig. 2 as an example we can observe the scores plot for the OPLS-DA model derived from CE-UV data. The control and infected mice in the last week of infection are shown in black boxes and red triangles respectively.

Fig. 2.

Scores plot for the OPLS-DA model derived from CE-UV data. The control and infected mice in the last week of infection are shown in black boxes and red triangles respectively. R² is the percentage of data explained by the model (near 95%) and Q² is the estimation of a new data point/sample being predicted correctly (78%). (Permission granted)

Using the same data, an autocorrelation algorithm was devised by the Barbas' group (Angulo et al. Reference Angulo, Garcia-Perez, Legido-Quigley and Barbas2009), this enabled finding further relationships between metabolites. The method was similar to STOCSY, a method applied previously in NMR data and based on the Pearson's correlation (Holmes et al. Reference Holmes, Cloarec and Nicholson2006). It was found that various metabolites of the phenylalanine cycle were indeed correlated and over metabolized in the infected animals. Another statistical correlation application but this time hyphenating two different analytical techniques, NMR and CE shown in 2D plots was published (Garcia-Perez et al. Reference Garcia-Perez, Couto Alves, Angulo, Li, Utzinger, Ebbels, Legido-Quigley, Nicholson, Holmes and Barbas2010). A number of metabolites, such as 3-ureidopropionate, p-cresol glucuronide, phenylacetylglycine (PAG), indoxyl sulfate, isocitrate, and trimethylamine, were identified as differentiating between infected and control animals. It was concluded by the authors that this correlation provided deeper mechanistic insight into the biological process in the infection. In Table 1 a list of the metabolites identified as important metabolites in the infection process with CE and NMR is shown.

Table 1.

The main metabolites found following a metabonomics approach using two different analytical techniques, CE and NMR

Ultra-performance liquid chromatography, a technique which has seen wide application in metabonomics in recent years and is preferred over conventional liquid chromatography, has been utilised to fingerprint the urine of infected mice. In Fig. 3 we observe what are called time trajectories, which are the scores mean trajectory derived from PCA using UPLC-MS data, where each point is a day in the life of the mice representing the mean position for the whole group during the course of the disease. In day 49 of infection, a significant deviation from the metabolic trajectory of controls is observed. This difference was attributed to PAG which was a metabolite related to microbial composition/metabolism and had been observed in previously published NMR and CE studies (Garcia-Perez et al. Reference Garcia-Perez, Whitfield, Bartlett, Angulo, Legido-Quigley, Hanna-Brown and Barbas2008 b; Wang et al. Reference Wang, Zhou, Vounatsou, Chen, Utzinger, Yang, Steinmann and Wu2008 a).

Fig. 3.

Scores mean trajectory derived from PCA of UPLC-MS data: Each point is a day in the life of the mice, representing the mean position for the whole group along the illness. (Ahead of publication)