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Identification of Glycosomes and Metabolic End Products in Pathogenic and Nonpathogenic Strains of Cryptobia Salmositica (Kinetoplastida: Bodonidae)

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Identification of Glycosomes and Metabolic End Products in Pathogenic and Nonpathogenic Strains of Cryptobia Salmositica (Kinetoplastida: Bodonidae) DISEASES OF AQUATIC ORGANISMS Vol. 42: 41–51, 2000 Published August 10 Dis Aquat Org Identification of glycosomes and metabolic end products in pathogenic and nonpathogenic strains of Cryptobia salmositica (Kinetoplastida: Bodonidae) B. F. Ardelli, J. D. S. Witt, P. T. K. Woo* Department of Zoology, University of Guelph, Guelph, Ontario N1G 2W1, Canada ABSTRACT: Whole cell lysates of pathogenic and nonpathogenic strains of Cryptobia salmositica were subjected to subcellular fractionation using differential and isopycnic centrifugation in sucrose. The glycolytic enzymes hexokinase, fructose-1,6-biphosphate aldolase, triosephosphate isomerase, glucosephosphate isomerase and glyceraldehyde-3-phosphate-dehydrogenase and the peroxisomal enzyme catalase were associated with a microbody that had a buoyant density in sucrose of 1.21 g cm–3. Lactate dehydrogenase was detected in whole cell lysates, but not in purified organelles. A microbody with a positive reaction for catalase was detected in electron microscope sections of the pathogenic and nonpathogenic strains. These catalase-containing microbodies fused with lipid bodies and vacuoles, arose by division from pre-existing microbodies and expelled their contents into the cytoplasm of the cell. Both strains also modified the catalase content in their microbodies. Under aerobic conditions, they metabolized glucose to pyruvate and lactate. We conclude that part of the glycolytic pathway in C. salmositica is compartmentalized in a microbody called the glycosome. KEY WORDS: Cryptobia · Glycosome · Glycolytic enzymes · Catalase · Pyruvate · Lactate Resale or republication not permitted without written consent of the publisher INTRODUCTION Li & Woo 1995) and in comparative studies aimed at a better understanding of the disease mechanism (Zuo & Cryptobia salmositica is a haemoflagellate which Woo 1996, 1997). Although C. salmositica causes mor- infects Oncorhynchus spp. on the west coast of North bidity and mortality in the economically important America (Woo & Poynton 1995) and is considered a Oncorhynchus spp. and results in considerable loss lethal pathogen in semi-natural and intensive salmon in some west coast hatcheries, we know little of the culture facilities (Bower & Margolis 1984). The parasite biology of this pathogen. is transmitted indirectly by the bite of blood sucking Cryptobia salmositica consumes oxygen (Thomas et aquatic leeches in freshwater streams and marine estu- al. 1992) and utilizes glucose and L-glutamine under aries (Becker & Katz 1965) or directly between fishes in vitro conditions (Li & Woo 1991). Prolonged expo- under laboratory and hatchery conditions (Woo & Weh- sure to sodium azide, a potent inhibitor of oxidative nert 1983). The pathogenic C. salmositica was attenu- phosphorylation, reduces oxygen consumption and ated by continuous in vitro culture (Woo& Li 1990); the activity, but does not kill the parasite (Thomas et al. resulting strain is infective but does not cause disease 1992). The recovery after removal of sodium azide is in fish (Li & Woo 1995). This attenuated strain of C. highly unusual because the latter normally kills aero- salmositica has been used successfully as a live vac- bic organisms within minutes of exposure. Since both cine (Ardelli et al. 1994, Sitja-Bobadilla & Woo 1994, C. salmositica strains (pathogenic and nonpathogenic) consume oxygen and glucose under in vitro conditions, they likely utilize the Embden-Meyerhof pathway for *Corresponding author. E-mail: [email protected] the production of ATP. However Thomas et al. (1992) © Inter-Research 2000 42 Dis Aquat Org 42: 41–51, 2000 were unable to detect 3 key enzymes of carbohydrate tion (10 700 × g for 10 min) and washed 5 times in cold and oxidative metabolism, namely hexokinase and tris-EDTA buffer (pH 7.8) containing 0.25 M sucrose, pyruvate kinase of glycolysis and cytochrome c oxi- 25 mM Tris-HCl and 1 mM EDTA. Cells were dis- dase of oxidative phosphorylation. rupted by incubation (1 h at 4°C) in a 0.03% solution of Members of the Kinetoplastida have an unusual Triton-X 100 and then disrupted cells were suspended organelle, the glycosome. It is bounded by a single in 25 ml of Tris-EDTA buffer. This suspension was membrane and contains 7 glycolytic enzymes, from centrifuged at 1310 × g for 10 min and the sediment hexokinase to phosphoglycerate kinase, which in other removed. The supernatant was centrifuged at 4530 × g cells are cytoplasmic (Opperdoes & Borst 1977). The for 10 min, the sediment removed and the supernatant Kinetoplastida are the only known living organisms further centrifuged at 42 900 × g for 20 min. The pellet where glycolysis is compartmentalized. The glyco- was resuspended in 60 ml of Tris-EDTA buffer and somes may also contain enzymes of peroxide metabo- 10 ml aliquots were layered on 10–60% (w/w) linear lism (Opperdoes 1988). It is assumed that the compart- sucrose gradients dissolved in 25 mM of Tris-HCl mentalization of glycolysis may facilitate the extremely (pH 7.2) and 1 mM of EDTA. A series of sucrose stan- high rate of glycolysis in the Kinetoplastida, particu- dards containing various concentrations of sucrose in larly in bloodforms of Trypanosoma brucei (Opperdoes Tris-EDTA buffer were prepared and the refractive 1995). indices (at 589 nm) were measured at 20°C (Price We hypothesize that Cryptobia salmositica survives 1982). The corresponding density was determined by prolonged exposure to sodium azide because it is able comparing the refractive index of standards with val- to switch between aerobic respiration and anaerobic ues computed by Eikenberry (see Price 1982). glycolysis. This switch may be accomplished, in part, Gradients were centrifuged (60 000 × g for 90 min) in because glycolysis is compartmentalized. To gain a an ultracentrifuge equipped with a swinging-bucket better understanding of carbohydrate and energy rotor. After centrifugation, each sucrose gradient was metabolism in C. salmositica, the objectives of the removed by puncturing the tube and extracting the present study were to (1) identify glycosomes and contents with a syringe. Next, gradients were cen- (2) determine some of the end products of glucose trifuged to pellet the contents, and pellets were pre- metabolism. pared for transmission electron microscopy (see below) to determine the separation of cell organelles within each gradient. Microbodies were determined by a MATERIALS AND METHODS catalase-positive reaction (see below). The gradient containing catalase-positive organelles was further In vitro culture of Cryptobia salmositica. A cloned purified by passaging twice through sucrose gradients. strain of pathogenic C. salmositica was used to infect The purified organelles were ruptured and prepared rainbow trout Oncorhynchus mykiss held at 11°C. The for electrophoresis to determine enzyme profiles. strain was initially isolated from Piscicola salmositica Isozyme electrophoresis. Thomas et al. (1992) could and details of the cloning of the parasite and fish main- not detect the enzymatic activity of hexokinase and tenance have been described earlier (Woo 1979). Blood pyruvate kinase. The same experiment was repeated from an infected trout was aseptically inoculated into in the present study and similar results were obtained. sterile culture flasks containing Minimum Essential Studies of another pathogen (Opperdoes et al. 1988) Medium (MEM) supplemented with Hanks’s salts, indicate the glycolytic enzymes of Cryptobia spp. have L-glutamine, 25 mM Hepes buffer, and 25% heat-inac- low activity. As a result, electrophoresis was used in tivated foetal bovine serum, and cultured. Short-term the present study to detect specific enzymes. This culture of the pathogenic strain does not affect the technique is sensitive and requires only small aliquots pathogenicity (Woo & Thomas 1992). The nonpatho- of the sample. Purified organelles (see above) were genic vaccine strain of C. salmositica was cloned and homogenized in Tris-EDTA buffer and centrifuged at has been maintained in MEM since 1989 (Woo & Li 4°C to sediment organelle membranes. The super- 1990). This nonpathogenic strain was reinoculated into natant was applied to cellulose acetate gels. Samples laboratory-raised chinook salmon Oncorhynchus tsha- were electrophoresed in a Tris-glycine buffer (pH 8.5) wytscha and serially passaged monthly for 8 mo (G. at 200 V for 15 min and stained for glyceraldehyde- Staines pers. comm.). It was also reisolated and main- phosphate dehydrogenase (EC 1.2.1.12), glucosephos- tained in MEM (present study). phate isomerase (EC 5.3.1.9), hexokinase (EC 2.7.1.1) Isolation and purification of microbodies (Opper- and lactate dehydrogenase (EC 1.1.1.27) as described does et al. 1984). Microbodies were isolated using a by Hebert & Beaton (1993), and catalase (EC 1.11.1.6), combination of differential and isopycnic centrifuga- fructose-1,6-biphosphate aldolase (EC 4.1.2.13) and tion. Briefly, parasites were harvested by centrifuga- triosephosphate isomerase (EC 5.3.1.1) as described by Ardelli et al.: Glycolysis in Cryptobia salmositica 43 Harris & Hopkinson (1976). Catalase activity was de- 30 pellet sections corresponding to a gold interference tected using 12.5% starch gels (Shaw & Prasad 1970). colour were discarded from each sample; the next 5 Preparation and fixation for transmission electron sections were collected on uncoated, copper-mesh microscopy. To isolate parasites from the blood of fish, grids. This was done for both pathogenic and nonpath- 10 to 15 drops of heat-inactivated horse serum were ogenic strains of Cryptobia salmositica such that 5 added to a heparinized blood sample (1.0 ml) to agglu- grids contained 5 pellet sections. Sections attached to tinate the red blood cells. The sample was centrifuged grids were stained as described and examined using a at 100 × g for 5 min to sediment most of the aggluti- JEOL 100CX electron microscope at 80 kV. Micro- nated cells with the parasites still in the supernatant. bodies were quantified by counting 10 parasite sec- Parasites from cultures were concentrated by centrifu- tions, in each of the 5 pellet sections, for a total of 250 gation in a cold (4°C) centrifuge at 6860 × g for 15 min. parasite sections.
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