31P-NMR studies of the metabolisms of the parasitic helminths Ascaris suum and Fasciola hepatica

31P-NMR has been applied to the study of the metabolisms of the intact parasitic helminths Ascaris suum (the intestinal roundworm) and Fasciola hepatica (the liver fluke). After calibration of the chemical shift of Pi in muscle extracts the internal pH of adult Ascaris worms and the effect of the pH of the external medium on the organism's internal pH were measured. Assignments of nearly all of the observable 31P resonances could be made. A large resonance from glycerophosphorylcholine whose function is unclear was observed but no signals from energy storage compounds such as creatine phosphate were detected. The profiles of the phosphorus-containing metabolites in both organisms were monitored as a function of time. Changes in sugar phosphate distributions but not ATP/ADP were observed. Studies of the drug closantel on Fasciola hepatica were performed. Initial effects of the drug were a decrease in glucose 6-phosphate and an increase in Pi with no substantial change in ATP levels as observed by 31P-NMR. Studies involving treatment with closantel followed by rapid freezing, extraction, and analytical determination of glycolytic intermediates confirmed NMR observations. This NMR method can serve as a simple noninvasive procedure to study parasite metabolism and drug effects on metabolism.     

Spatiotemporal and gender‐specific parasitism in two species of gobiid fish

Parasitism is considered a major selective force in natural host populations. Infections can decrease host condition and vigour, and potentially influence, for example, host population dynamics and behavior such as mate choice. We studied parasite infections of two common marine fish species, the sand goby (Pomatoschistus minutus) and the common goby (Pomatoschistus microps), in the brackish water Northern Baltic Sea. We were particularly interested in the occurrence of parasite taxa located in central sensory organs, such as eyes, potentially affecting fish behavior and mate choice. We found that both fish species harbored parasite communities dominated by taxa transmitted to fish through aquatic invertebrates. Infections also showed significant spatiotemporal variation. Trematodes in the eyes were very few in some locations, but infection levels were higher among females than males, suggesting differences in exposure or resistance between the sexes. To test between these hypotheses, we experimentally exposed male and female sand gobies to infection with the eye fluke Diplostomum pseudospathaceum. These trials showed that the fish became readily infected and females had higher parasite numbers, supporting higher susceptibility of females. Eye fluke infections also caused high cataract intensities among the fish in the wild. Our results demonstrate the potential of these parasites to influence host condition and visual abilities, which may have significant implications for survival and mate choice in goby populations.     

Phosphofructokinase from Fasciola hepatica: activation by phosphorylation and other regulatory properties distinct from the mammalian enzyme

Phosphofructokinase from the liver fluke, Fasciola hepatica, was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase isolated from this organism. Phosphorylated fluke phosphofructokinase had a sevenfold lower apparent Km for its substrate, Fru-6-P, and an eightfold higher 0.5 Vopt for ATP, the enzyme's primary inhibitor, than native phosphofructokinase. Activation of fluke phosphofructokinase following phorphorylation by a mammalian protein kinase catalytic subunit was previously reported (E. S. Kamemoto and T. E. Mansour (1986) J. Biol. Chem. 261, 4346-4351). The catalytic subunit of protein kinase isolated from the liver fluke phosphorylated sites on fluke phosphofructokinase similar to those phosphorylated by the mammalian enzyme. Maximal phosphate incorporation was 0.3 mol P/mol of protomer. The native enzyme was found to contain 1.3 mol P/mol of protomer. In contrast to fluke phosphofructokinase, activity of the mammalian heart enzyme was slightly decreased following phosphorylation. The dependence of allosteric interaction on an acidic pH observed with the mammalian phosphofructokinase was not observed with the fluke enzyme. Unlike mammalian phosphofructokinase, allosteric kinetics of the fluke enzyme was observed at alkaline pH (8.0). Fluke phosphofructokinase was found to be relatively insensitive to inhibition by citrate, a known potent inhibitor of the mammalian enzyme. Fru-2,6-P2, a potent modifier of phosphofructokinase from a variety of sources, was found to activate both native and phosphorylated fluke phosphofructokinase. The most potent activators of fluke phosphofructokinase were found to be Fru-2,6-P2, AMP, and phosphorylation. The endogenous level of Fru-2,6-P2 in the flukes was determined to be 29 +/- 1.3 nmol/g wet wt, a level that may well modulate enzyme activity. Fru-6-P,2-kinase, the enzyme responsible for synthesis of Fru-2,6-P2, was found to be present in the flukes. Our results suggest physiological roles for phosphorylation and Fru-2,6-P2 in regulation of fluke phosphofructokinase.     

Genotype-Specific vs. Cross-Reactive Host Immunity against a Macroparasite

Vertebrate hosts often defend themselves against several co-infecting parasite genotypes simultaneously. This has important implications for the ecological dynamics and the evolution of host defence systems and parasite strategies. For example, it can drive the specificity of the adaptive immune system towards high genotype-specificity or cross-reactivity against several parasite genotypes depending on the sequence and probability of re-infections. However, to date, there is very little evidence on these interactions outside mammalian disease literature. In this study we asked whether genotype-specific or cross-reactive responses dominate in the adaptive immune system of a fish host towards a common macroparasite. In other words, we investigated if the infection success of a parasite genotype is influenced by the immunization genotype. We reciprocally immunized and re-exposed rainbow trout (Oncorhynchus mykiss) to a range of genotypes of the trematode eye fluke Diplostomum pseudospathaceum, and measured infection success of the parasite. We found that the infection success of the parasite genotypes in the re-exposure did not depend on the immunization genotype. While immunization reduced average infection success by 31%, the reduction was not larger against the initial immunization genotype. Our results suggest significant cross-reactivity, which may be advantageous for the host in genetically diverse re-exposures and have significant evolutionary implications for parasite strategies. Overall, our study is among the first to demonstrate cross-reactivity of adaptive immunity against genetically diverse macroparasites with complex life cycles.     

Composition and metabolism of phospholipids of Fasciola hepatics, the common liver fluke.

1. The phospholipid composition of Fasciola hepatica, the common liver fluke, was compared to that of the liver of the host animals (rats and cattle). Considerable differences were found:monoacyl-sn-glycero-3-phosphorylcholine, hardly detectable in the liver, was found in significant amounts in the parasite. On the other hand, sphingomyelin, a normal constituent in the liver, appears to be absent in the liver fluke. Fasciola hepatica isolated from rat and cow liver had a strikingly similar phospholipid composition. 2. Qualitative and quantitative differences were also found between the fatty acyl constituents of the phospholipids of the parasite and the liver. The major difference was the presence of eicosaenoic and eicosadienoic acids in the parasite, whereas these acids were not detected in the liver. 3. In vitro incubations of Fasciola hepatica in the presence of (32P)phosphate and (2-3H)glycerol resulted in the labelling of all phospholipids of the parasite, except that the 3H label did not incorporate into ethanolamine plasmalogen. This is in agreement with the concept that in animals, glycerol is introduced into plasmalogens via dihydroxyacetonephosphate. 4. Homogenates of liver flukes were found to catalyze the synthesis of phosphatidylcholine from 1,2-diacyl-sn-glycerols and CDPcholine. 5. These results strongly suggest that Fasciola hepatica is capable of synthesizing at least part of its fatty acids and phospholipids.     

Peptide synthesis by recombinant Fasciola hepatica cathepsin L1

Synthesis of the tripeptide Z-Phe-Arg-SerNH2 has been accomplished by a recombinant cysteine protease, cathepsin L1 from liver fluke (Fasciola hepatica), using Z-Phe-Arg-OMe as acyl acceptor and SerNH2 as nucleophile in 0.1 M ammonium acetate pH 9.0-12.5% v/v acetonitrile at 37 degrees C. LC-MS detection indicated tripeptide formation after 10 min, continuing up to 5.5 h. The ester Z-Phe-Arg-OMe was detected throughout the experiment but the hydrolysis product Z-Phe-Arg-OH appeared early and in quite large amounts. We believe that this is the first application of a parasite protease in enzymatic peptide synthesis.     

Variation in parasite resistance of Arctic charr,Salvelinus alpinus,between and within sympatric morphs

Genetic variation in resistance against parasite infections is a predominant feature in host–parasite systems. However, mechanisms maintaining genetic polymorphism in resistance in natural host populations are generally poorly known. We explored whether differences in natural infection pressure between resource‐based morphs of Arctic charr (Salvelinus alpinus) have resulted in differentiation in resistance profiles. We experimentally exposed offspring of two morphs from Lake Þingvallavatn (Iceland), the pelagic planktivorous charr (“murta”) and the large benthivorous charr (“kuðungableikja”), to their common parasite, eye fluke Diplostomum baeri, infecting the eye humor. We found that there were no differences in resistance between the morphs, but clear differences among families within each morph. Moreover, we found suggestive evidence of resistance of offspring within families being positively correlated with the parasite load of the father, but not with that of the mother. Our results suggest that the inherited basis of parasite resistance in this system is likely to be related to variation among host individuals within each morph rather than ecological factors driving divergent resistance profiles at morph level. Overall, this may have implications for evolution of resistance through processes such as sexual selection.     

Determination of closantel residues in plasma and tissues by high-performance liquid chromatography with fluorescence detection

The influence of the pH of the mobile phase with some modifiers on the chromatographic behavior and fluorescence properties of closantel have been investigated. At acidic pH values (2–6), the benzamide moiety of the closantel forms a six-membered ring by hydrogen bonding and possesses a native fluorescence. Using the fluorescence emission of closantel at λ_ex=335 nm, λ_em=510 nm, and pH 2.5 of the mobile phase, a linear calibration curve was estimated over a concentration range of about two orders of magnitude with a correlation coefficient larger than 0.992. The limit of the fluorescence detection was 10 μg/kg. This value was at least 10 times lower than that using UV detection. The method was applied to the determination of closantel in plasma and tissue samples, purified by a solid-phase extraction with C₁₈ cartridges.     

Alteration of bacterial surface electrostatic potential and pH upon adhesion to a solid surface and impacts to cellular bioenergetics

In our previous study [Hong Y, Brown DG (2009) Appl Environ Microbiol 75(8):2346–2353], the adenosine triphosphate (ATP) level of adhered bacteria was observed to be 2–5 times higher than that of planktonic bacteria. Consequently, the proton motive force (Δp) of adhered bacteria was approximately 15% greater than that of planktonic bacteria. It was hypothesized that the cell surface pH changes upon adhesion due to the charge‐regulated nature of the bacterial cell surface and that this change in surface pH can propagate to the cytoplasmic membrane and alter Δp. In the current study, we developed and applied a charge regulation model to bacterial adhesion and demonstrated that the charge nature of the adhering surface can have a significant effect on the cell surface pH and ultimately the affect the ATP levels of adhered bacteria. The results indicated that the negatively charged glass surface can result in a two‐unit drop in cell surface pH, whereas adhesion to a positively charged amine surface can result in a two‐unit rise in pH. The working hypothesis indicates that the negatively charged surface should enhance Δp and increase cellular ATP, while the positively charged surface should decrease Δp and decrease ATP, and these results of the hypothesis are directly supported by prior experimental results with both negatively and positively charged surfaces. Overall, these results suggest that the nature of charge on the solid surface can have an impact on the proton motive force and cellular ATP levels. Biotechnol. Bioeng. 2010;105: 965–972. © 2009 Wiley Periodicals, Inc.     

Effects of ATP on the intermediary steps of the reaction of the (Na+ + K+)-ATPase. IV. Effect of ATP on K0.5 for Na+ and on hydrolysis at different pH and temperature.

The pH optimum for (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) depends on the combination of monovalent cations, on the ATP concentration and on temperature. ATP decreases the Na+ concentration necessary for half maximum activation, K0.5 for Na+ (Na+ + K+ = 150 mM), and the effect is pH and temperature dependent. At a low ATP concentration a decrease in pH leads to an increase in K0.5 for Na+, while at the high ATP concentration it leads to a decrease. K0.5 for ATP for hydrolysis decreases with an increase in pH. The fractional stimulation by K+ in the presence of Na+ decreases with the ATP concentration, and at a low ATP concentration K+ becomes inhibitory, this being most pronounced at 0 degrees C. The results suggest that (a) ATP at a given pH has two different effects: it increases the Na+ relative to K+ affinity on the internal site (K0.5 for ATP at pH 7.4, 37 degrees C, is less than 10 microM); it increases the molar activity in the presence of Na+ + K+ (K0.5 for ATP at pH 7.4, 37 degrees , is 127 microM), (b) binding of the cations to the external as well as the internal sites leads to pK changes (Bohr effect) which are different for Na+ and for K+, i.e. the selectivity for Na+ relative to K+ depends both on ATP and on the degree of protonation of certain groups on the system, (c) ATP involves an extra dissociable group in the determination of the selectivity of the internal site, and thereby changes the effect of an increase in protonation of the system from a decrease to an increase in selectivity for Na+ relative to K+.     

Protein tyrosine kinase activity in human malaria parasite Plasmodium falciparum.

Protein tyrosine kinases (PTKs) are believed to be implicated in the parasite growth, maturation and differentiation functions. Protein tyrosine kinase activity was found to be distributed in all the stages of P. falciparum parasite maturation. Membrane bound PTK activity was found to be increased during maturation process (ring stage to trophozoite stage) in chloroquine sensitive strains. In vivo conversion of the schizont stage to ring stage via release of merozoites was associated with a decrease in PTK activity. Chloroquine inhibited the membrane bound PTK activity in a dose dependent manner (IC50 = 45 microM). Kinetic studies show that chloroquine is a competitive inhibitor of PTK with respect to peptide substrate and noncompetitive with respect to ATP indicating that chloroquine inhibits PTK activity by binding with protein substrate binding site. The results suggest that maturation of malaria parasite is related to PTK and inhibition of this activity by chloroquine could provide a hypothesis to explain the mechanism of action of chloroquine.     

The draft genome of the carcinogenic human liver fluke Clonorchis sinensis

Background

Clonorchis sinensis is a carcinogenic human liver fluke that is widespread in Asian countries. Increasing infection rates of this neglected tropical disease are leading to negative economic and public health consequences in affected regions. Experimental and epidemiological studies have shown a strong association between the incidence of cholangiocarcinoma and the infection rate of C. sinensis. To aid research into this organism, we have sequenced its genome.

Results

We combined de novo sequencing with computational techniques to provide new information about the biology of this liver fluke. The assembled genome has a total size of 516 Mb with a scaffold N50 length of 42 kb. Approximately 16,000 reliable protein-coding gene models were predicted. Genes for the complete pathways for glycolysis, the Krebs cycle and fatty acid metabolism were found, but key genes involved in fatty acid biosynthesis are missing from the genome, reflecting the parasitic lifestyle of a liver fluke that receives lipids from the bile of its host. We also identified pathogenic molecules that may contribute to liver fluke-induced hepatobiliary diseases. Large proteins such as multifunctional secreted proteases and tegumental proteins were identified as potential targets for the development of drugs and vaccines.

Conclusions

This study provides valuable genomic information about the human liver fluke C. sinensis and adds to our knowledge on the biology of the parasite. The draft genome will serve as a platform to develop new strategies for parasite control.     

Spatially resolved changes in diabetic rat skeletal muscle metabolism in vivo studied by 31P-n.m.r. spectroscopy.

Phase-modulated rotating-frame imaging (p.m.r.f.i.), a localization technique for 31P-n.m.r. spectroscopy, has been applied to obtain information on the heterogeneity of phosphorus-containing metabolites and pH in the skeletal muscle of control and streptozotocin-diabetic rats. Using this method, the metabolic changes in four spatially resolved longitudinal slices (where slice I is superficial and slice IV is deep muscle) through the ankle flexor muscles have been investigated at rest and during steady-state isometric twitch-contraction at 2 Hz. At rest, intracellular pH was lower, and phosphocreatine (PCr)/ATP was higher, throughout the muscle mass in diabetic compared with control animals. The change in PCr/ATP in diabetic muscle correlated with a decrease in the chemically determined ATP concentration. During the muscle stimulation period, the decrease in pH observed in diabetic muscle at rest was maintained, but not exacerbated, by the contractile stimulus. Stimulation of muscle contraction caused more marked changes in PCr/(PCr + Pi), PCr/ATP and Pi/ATP in the diabetic group. These changes were most evident in slice III, which contains the greatest proportion of fast glycolytic-oxidative (type IIa) fibres, in which statistically significant differences were observed for all metabolite ratios. The results presented suggest that some degree of heterogeneity occurs in diabetic skeletal muscle in vivo with respect to the extent of metabolic dysfunction caused by the diabetic insult and that regions of the muscle containing high proportions of type IIa fibres appear to be most severely affected.     

Phosphofructokinase in the liver fluke Fasciola hepatica. Purification and kinetic changes by phosphorylation

Phosphofructokinase from the liver fluke Fasciola hepatica was purified from extracts of the whole organisms. The molecular weight of the protomer as determined by sodium dodecyl sulfate-gel electrophoresis is 83,000. Phosphorylation of the liver fluke phosphofructokinase by the catalytic subunit of cAMP-dependent protein kinase occurred at a rate that was at least an order of magnitude greater than that observed with mammalian heart phosphofructokinase. The maximum level of phosphate incorporated was 0.22 mol P/mol of protomer. The kinetic properties of the enzyme were greatly altered as a result of phosphorylation. Compared to native enzyme, phosphorylated enzyme had a greater affinity for its substrate Fru-6-P and a decreased sensitivity to inhibition by ATP. These kinetic changes were similar to those of native enzyme in the presence of positive modifiers such as AMP. AMP also activated the phosphorylated enzyme. Activation of the phosphorylated enzyme by AMP was characterized by a further increase in affinity for Fru-6-P and a further decrease in sensitivity to ATP inhibition. Thus, the liver fluke phosphofructokinase can be modulated by covalent phosphorylation as well as noncovalent binding of different modifier ligands.     

31P-NMR study of resting in vitro rat diaphragm exposed to hypercapnia

We have reported previously that, when exposed to hypercapnia of various intensities, the diaphragm reduces its force of twitch and tetanic contractions in the in vitro rat preparation as well as in the in vivo dog preparation. The experiments reported here with 31P nuclear magnetic resonance (31P-NMR) spectroscopy attempt to examine cellular mechanisms that might be responsible for this deterioration in mechanical performance. Specifically they describe certain characteristics of this preparation and cautions needed to study the resting in vitro rat diaphragm with such techniques. Second, they report the response of intracellular pH (pHi), phosphocreatine (PCr), ATP, and inorganic phosphate (Pi) in the resting in vitro rat diaphragm exposed to long-term normocapnia or to long-term hypercapnia. The results show that 1) to maintain a viable preparation, it was necessary to keep the diaphragm extended to an area approximating that at functional residual capacity, 2) the diaphragm seemed quite capable of maintaining a constant pHi and constant contents of ATP and Pi during normocapnia, but there was a gradual decline in PCr, and 3) during hypercapnia there was a significant decrease in pHi, but the behavior of the phosphate metabolites was exactly as during normocapnia. The results suggest that the decrease in mechanical performance of the diaphragm is probably not due to a decrease in the availability of the high-energy phosphates, although they do not completely exclude this possibility or possibilities related to regional compartmentation.     

Interconversion of two distinct states of active CF0-CF1 (chloroplast ATPase complex) in chloroplasts

Chloroplast ATPase complex is activated by illumination in the presence or absence of dithiothreitol. ATPase complex which has been activated without dithiothreitol catalyzes ATP hydrolysis which is insensitive to stimulation by NH4Cl and is highly sensitive to medium pH. Addition of dithiothreitol during illumination results in an increase in the stimulating effect of NH4Cl on ATP hydrolysis and a decrease in pH sensitivity of ATP hydrolysis. With increasing time in the dark, the ability of NH4Cl to stimulate ATP hydrolysis decreases and the effect of pH on the ATP hydrolysis increases. The onset of resistance to NH4Cl stimulation and the increase in sensitivity to pH are accelerated by ADP and the acceleration is inhibited by Pi. ATP hydrolysis restores NH4Cl sensitivity and renders the activity more resistant to pH. These results suggest that active chloroplast ATPase complex converts its state reversibly from the NH4Cl-insensitive and highly pH-sensitive one to the NH4Cl-sensitive and relatively pH-insensitive one. The conversion from the former to the latter requires both sulfhydryl compound and energy.     

Tolerance of liver fluke infection varies between breeds and producers in beef cattle

Liver flukes (Fasciola spp.) are important helminth parasites of livestock globally and cause substantial reductions in health and productivity of beef cattle. Attempts to control fluke have been thwarted by the difficulty of vaccine design, the evolution of flukicide resistance and the need to control the intermediate snail host. Mechanisms to reduce the impact of parasites on animal performance have typically focused on promoting host resistance – defined as the ability of the host to kill and remove the parasite from its system – and such strategies include improving protein nutrition or selective breeding for resistance. Organisms, however, have another broad mechanism for mitigating the impact of parasites: they can show tolerance, defined as the ability to maintain health or performance under increasing parasite burden. Tolerance has been studied in the plant literature for over a century, but there are very few empirical studies of parasite tolerance in livestock. In this study, we used data collected from > 90 000 beef cattle to estimate the impact of the severity of liver fluke infection on performance and variation in tolerance of fluke. Severity of liver fluke infection was estimated using liver “fibrosis score” on a scale of 0–3 and performance estimated as (1) age at slaughter and (2) daily dead weight gain. Animals with higher fibrosis scores were slaughtered around 2 weeks later than animals with no fluke and gained around 10 g less weight per day. There was also considerable variation in these effects of fibrosis score, such that animals from different producers and breeds varied in their tolerance of fluke infection. While breeds did not vary in the association between fibrosis and age at slaughter, there was considerable variation among producers: high fibrosis score delayed slaughter by up to 50 days in some producers, but not at all in others. Meanwhile, there was support for variation in the slope of daily dead weight gain on fibrosis score among both breeds and producers, with some unaffected by high fluke scores and some breeds and producers experiencing a 20 g/day lower weight gain under high fluke scores. Our results point to the potential for both environmental and genetic variation in tolerance of liver fluke in cattle, paving the way for quantitative genetic and nutritional research into the feasibility of promoting tolerance as a disease mitigation strategy.     

Infection with the carcinogenic human liver fluke, Opisthorchis viverrini

Throughout Southeast Asia there is a strikingly high incidence of cholangiocarcinoma (CCA--hepatic cancer of the bile duct epithelium), particularly in people from rural settings in Laos and Northeast Thailand who are infected with the liver fluke, Opisthorchis viverrini, one of only three carcinogenic eukaryotic pathogens. More ubiquitous carcinogenic microbes, such as Helicobacter pylori, induce cancer in less than 1% of infected people, while as many as one-sixth of people with opisthorchiasis will develop CCA. The mechanisms by which O. viverrini causes cancer are multi-factorial, involving mechanical irritation from the activities and movements of the flukes, immunopathology, dietary nitrosamines and the secretion of parasite proteins that promote a tumourigenic environment. Genomic and proteomic studies of the liver fluke secretome have accelerated the discovery of parasite proteins with known/potential roles in pathogenesis and tumourigenesis, establishing a framework towards understanding, and ultimately preventing, the morbidity and mortality attributed to this highly carcinogenic parasite.     

Reversible decreases in ATP and PCr concentrations in anoxic turtle brain

A hallmark of anoxia tolerance in western painted turtles is relative constancy of tissue adenylate concentrations during periods of oxygen limitation. During anoxia heart and brain intracellular compartments become more acidic and cellular energy demands are met by anaerobic glycolysis. Because changes in adenylates and pH during anoxic stress could represent important signals triggering metabolic and ion channel down-regulation we measured PCr, ATP and intracellular pH in turtle brain sheets throughout a 3-h anoxic-re-oxygenation transition with ³¹P NMR. Within 30 min of anoxia, PCr levels decrease 40% and remain at this level during anoxia. A different profile is observed for ATP, with a statistically significant decrease of 23% occurring gradually during 110 min of anoxic perfusion. Intracellular pH decreases significantly with the onset of anoxia, from 7.2 to 6.6 within 50 min. Upon re-oxygenation PCr, ATP and intracellular pH recover to pre-anoxic levels within 60 min. This is the first demonstration of a sustained reversible decrease in ATP levels with anoxia in turtle brain. The observed changes in pH and adenylates, and a probable concomitant increase in adenosine, may represent important metabolic signals during anoxia.