Internakmd Journal for Parasitology Vol. 22. No. 8, pp. Primed in Grear Brifain
MANNITOL
I I57-I 163, I992
METABOLISM
WOJTEK P. MICHALSKI,* CSIRO
Division
of Animal
Health,
Animal
IN EIMERIA
TENELLA
JOHN A. EDGAR and STEPHEN Health
Research
Laboratory,
Parkville,
J. PROWSE Victoria
3052, Australia
(Received 3 February 1992; accepted 19 June 1992)
Ah&act-MICHALsKI W. P., EDGAR J. A. and PROWSES. J. 1992. Mannitol metabolism in Eimeria tenella. International Journalfor Parasitology 22: 1157-l 163. Unsporulated oocysts of Eimeria tenella contain large quantities of carbohydrates, namely amylopectin, mannitol and glucose. Analysis of the carbohydrate content of sporulating oocysts revealed that mannitol content increased markedly during early stages of sporogony (first 4-6 h) but slowly diminished during the next 40 h of sporulation. Accumulation of mannitol was accompanied by a rapid decrease in amylopectin and free glucose, suggesting that mannitol might be synthesized from glucose released from amylopectin. Mannitol was also detected in sporozoite and merozoite extracts. All four mannitol cycle enzymes were detected in oocysts. Sporozoites excysted in vitro had lower activities of all four enzymes. Mannitol-1-phosphatase and mannitol dehydrogenase activity was also detected in merozoites obtained from the second stage schizonts. Sporozoites incubated with ‘%glucose accumulated radioactively labelled precursor continuously for over 12 h and some of the ‘%-glucose was converted into %mannitol. These results indicate that mannitol plays an important role in the metabolism and development of the intracellular stages of the parasite. INDEX KEY sporozoites.
WORDS:
Mannitol;
carbohydrate
metabolism;
INTRODUCTION Coccrorosis is an economically important parasitic disease of poultry which resulted from infection by protozoa of the genus Eimeriu. One of the better characterized species, E. tenella, infects and destroys the epithelial cells lining the caeca resulting in high morbidity and mortality. Chickens are infected by coccidia following ingestion of the vegetative stage of the parasite, the sporulated oocyst. Infective sporozoites are released from oocysts in the intestine and rapidly invade epithelial cells of the caeca. Sporozoites then undergo several generations of asexual reproduction (merogony) before entering a stage of sexual differentiation leading to the formation of oocysts (oogony). Unsporulated oocysts are then shed in the faeces and sporulate when exposed to the appropriate levels of oxygen and humidity (Levine, 1982). Beyond these requirements the oocyst is entirely dependent on endogenous energy to support the process of sporulation. Amylopectin, a branched polysaccharide (polyglucose), is found in granules in almost all stages of the E. tenella life-cycle (Wang, Weppelman & Lopez-Ramos, 1975). Recently, it has been found that oocysts and sporozoites also contain mannitol previ-
Eimeriu
renellu; sporulating
oocysts;
ously reported exclusively in plants and bacteria (Lewis & Smith, 1967; Schmatz, Arison, Dashkevicz, Liesch & Turner, 1988; Schmatz, 1989). Many fungi contain mannitol and various theories have been proposed to explain its role, the most convincing of which is its use in the mannitol cycle (Hult & Gatenbeck, 1978, 1979). A similar mannitol cycle has recently been observed in E. tenella. All four mannitol cycle enzymes namely, mannitol-l-phosphate dehydrogenase (MlPD), mannitol-1-phosphatase (M-lPase), mannitol dehydrogenase (MDH) and hexokinase were found in cell-free extracts of unsporulated oocysts of E. tenella (Schmatz, Baginsky & Turner, 1989 and Fig. 1). It has not been established whether any of the other stages of the parasite possess mannitol cycle enzymes (Schmatz, 1989). At present, information on the function of the mannitol cycle in the coccidia is limited to the unsporulated oocysts and it has been postulated that mannitol is generated by the parasite in the gut during late oogenesis (Schmatz et al., 1988). In this paper we present evidence indicating that mannitol is also synthesized in sporulating oocysts and sporozoites from glucose. MATERIAL AND METHODS
* To whom all correspondence
should be addressed.
Oocystpreparation andsporulation. A line of E. tenella was established in our laboratory from a single oocyst obtained 1157
1158
W. P. MICHALSKI. J. A. EDGAR and S. J. PROWSE
Mannitol
Cycle
Glucose-&phosphate
QlUCO8~-6-P imMner~*e
It II
Fructose-&phosphate
Mannitol-l-phosphate
Fructose
Mannitol
FIG. 1. The mannitol cycle in Eimeria tenella. The main enzymes involved are: mannitol- l-phosphate dehydrogenase (M-I-PDH), mannitol-1-phosphatase (M-I-Pase), mannitol dehydrogenase (MDH) and hexokinase. Glucose is converted to fructose-dphosphate via hexokinase and glucose-6-phosphate isomerase. Fructose-6-phosphate can enter the mannitol cycle via M-I-PDH and is reduced to mannitol-l-phosphate which in turn is dephosphorylated by M-1-Pase to yield mannitol. Mannitol is then oxidized to fructose by MDH and is rephosphorylated to fructose-6phosphate by hexokinase. Note that M-I-PDH can also oxidase NADPH (Schmatz et al., 1989).
from a field sample. The line was passaged in SPF birds aged between 3 and 6 weeks. Oocysts were prepared from the caeca of infected birds as described by Prowse & Pallister (1989) except that mucosal homogenate was digested with trypsin at 41°C for only 2 h. Freshly prepared oocysts were resuspended in sterile phosphate buffer-saline (PBS) and immediately sparged with high purity nitrogen for 15 min and kept under anaerobic conditions at 4’C until needed. For the kinetic experiments they were used immediately. Sporulation was achieved by incubating 100 ml of the oocyst suspension (5 x lo6 ml-‘) in a 500 ml flask in a 28°C waterbath at 100 beats min-‘. Samples of sporulating oocysts (3 x 10’) were collected every 2 h under sterile conditions for mannitol extraction. They were then sparged with nitrogen and snapfrozen in liquid nitrogen. Isolation of sporocysts. Fractions enriched in sporocysts were obtained as follows: oocysts sporulated for 12, 24, 36 and 48 h were fractured by mixing oocysts with glass beads on
a vortex mixer. Mixing was continued (approximately 8-10 min) until no oocysts were detected under the microscope. Homogenates containing mostly sporocysts and cell debris were washed twice in PBS and examined under the microscope. These fractions enriched in sporocysts contained no oocysts or sporozoites and were used immediately for mannitol extraction. Isolation of sporozoites and merozoiies. Sporozoites were obtained from oocysts sporulated for 24, 36 and 48 h using the method described previously (Prowse & Pallister, 1989). The sterile sporozoite preparations were either used immediately in enzyme activity assay or pelleted and snapfrozen in liquid nitrogen for mannitol extraction. For merozoite isolation, chickens were inoculated with 0.5-l .O x 10’ oocysts and the caeca removed 113 h later. They were slit open and washed in cold PBS until clean. The mucosa was scraped off and homogenized in a Waring blendor for 20 s in PBS containing 2 mg ml-’ glucose. The homogenate was transferred to a tube and the clumps allowed to settle. The supernatant was filtered through eight layers of gauze and centrifuged at 15OOxg for 10 min. The pellet was then resuspended in 50 ml of cold lysis buffer containing 8.3 g I-‘NH,Cl,0.372 g I-‘EDTA,l g 1.‘K&O, pH 7.6. After 30 min on ice the suspension was diluted in 2 vol. of PBS/glucose and centrifuged at 1500 x g for 10 mm and suspended in 30 ml of PBS/glucose. The suspension was then passed down a nylon wool column containing 1.6 g of washed nylon wool in a 10 ml syringe. The merozoites were washed off the column with PBS, centrifuged as above and counted. On average the merozoite preparations contained approximately IO-15% epithelial cells. Epithelial cells were obtained from the mucosa of uninfected chickens in the same manner as described for merozoite preparation, except that the cells were not passed down a nylon wool column. Preparation of crude enzyme. Crude enzyme homogenates were obtained from unsporulated (10’) and sporulated (5 x 10’) oocysts using the method described previously (Schmatz et al., 1989). Crude enzyme preparations from sporozoites (10’) and merozoites (108)were also obtained using the same method except that the cells were homogenized by sonication (MSE Soniprep, 3 x 10 s at 70% power output) at 4°C. Crude homogenates from epithelial cells of uninfected chickens were also prepared by sonication. All homogenates were desalted on a Bio-Gel P-6DG column before the final concentration step. Oocyst homogenates contained approximately 8-10 mg protein ml-‘, whereas the preparations from sporozoites and merozoites contained 2-3 mg protein ml ’ Assays for enzyme activities. All enzyme assays were initiated with substrate and monitored for 8 min unless stated otherwise. Determinations were made in 1 ml cuvettes by continuous scanning against appropriate reaction blanks. Dehydrogenase activities were determined by measuring coenzyme reduction or oxidation spectrophotometricaily at 340 nm in a Hitachi U-2000 spectrophotometer. Specific activity of these enzymes is expressed in pmoles of nicotinamide adenine nucleotides reduced or oxidized per min (one unit) per mg of protein. The activity of mannitol-lphosphatase is expressed in pmoles of phosphate released per min (one unit) per mg of protein. Manniiol- 1-phosphate dehydrogenase (M- I-PDH, EC
Mannitol
cycle in E. tenella
1.1.1.17). The enzyme activity was measured at 25°C in a reaction mixture containing 50 mM-HEPES @H 7.5), 1 mMEDTA, 1 mM-NAD (P)H, 2.5 mM-fructose-6-phosphate (F6-P) and the crude enzyme preparation. The reverse reaction was determined at 25°C in a reaction mixture containing 50 mM-3-( 1, I-dimethyl-2-hydroxyethyl)-2-hydroxypropane sulphonic acid (AMPSO; pH 9.5), 1 mM-EDTA, 2 mM-NADP, 50 mM-mannitol-l-phosphate (M-l-P) and the crude enzyme. Mannitol-I-phosphatase (M-I-Pase, EC 3.1.3.22). The enzyme was assayed at 25°C in 50 mM-HEPES (pH 7.0) containing 1 mM-mannitol-l-phosphate, 1 mM-EDTA and crude enzyme. Free phosphate released during the 15 min incubation was determined using the Ames reagent (Ames, 1966). Mannirol dehydrogenase (MDH, EC 1.1.1.67). MDH activity was determined at 25°C in a reaction mixture containing 50 mM-AMPS0 (pH 9.0), 1 mM-EDTA, 3.0 mMNAD, 50 mM-mannitol and the crude enzyme preparation. Hexokinase (EC 2.7.1.1). Hexokinase was assayed at 25°C in a coupled reaction system as described by Schmatz et al. (1989). The reaction mixture contained 50 mM-HEPES (pH 7.5) 5 mM-MgCl,, 2 mh+ATP, 0.25 mht-NADP, 5 mMfructose, glucose-6-phosphate dehydrogenase (10 units, Sigma G-5885), phosphoglucose isomerase (5 units, Sigma P5538) and the crude enzyme. In some assays, fructose was substituted by glucose to determine substrate specificity. Amylopectin and free glucose determination. Amylopectin granules were prepared from oocysts (2 x 106) according to the method of Wang er al. (1975). Amylopectin content was determined as the amount of glucose released after acid hydrolysis. Amylopectin samples were hydrolysed in glass ampules (sealed under nitrogen) with 0.6 N-HCl at 105’C for 2.5 h. Hydrolysates were then neutralized with 0.5 i+NaOH and the glucose concentration determined by the enzymatic method (glucose oxidase/peroxidase test kits, Sigma Diagnostics or Boehringer Mannheim Diagnostica) using a freshly prepared glucose solution as an external standard. Free glucose in supernatants of crushed oocysts was measured by the enzymatic method. Gas chromatography/mass spectrometry (GC/MS) analysis was used for glucose determination in mannitol extracts (see below). Mannitol extraction. Preparations of oocysts and sporocysts (both 3 x 10’) were resuspended in 1 ml of double distilled water and homogenized with glass beads. Sporozoites (3 x lo*), merozoites (5 x 10’) and epithelial cells (10”) were resuspended in double distilled water and homogenized by sonication (MSE Soniprep, 3 x 10 s at 70% power output). Mannitol extracts were prepared from the homogenates using the procedure described previously (Schmatz et al., 1988). Preparation of trimethylsilyl derivatives and chromarography. Extracted fractions and standards were derivatized by trimethylsilization (TMS). Dried extracts were dissolved in equal volumes of anhydrous pyridine and bis(trimethylsilyl)trifluoroacetamide (BSTFA, Sigma), and incubated for 12 h at 60°C. These TMS-derivatized samples were injected directly into the gas chromatography system. TMS-derivatized samples were analysed by GC/MS. GC/MS was carried out on a JOEL JMS-DX 303 mass spectrometer coupled to a Varian Vista 6000 gas chromatograph and a
1159
JOEL JMA-DA 5000 data system. The vitreous silica capillary column (25 m x 0.33mm ID) with BP1 (SGE) as the liquid phase (0.5 pm film) was connected directly to the ion source and temperature programmed from 180 to 280°C at either 10 or 4°C mini’. Electron impact mass spectra were generated using electrons at 70 eV and an accelerating voltage of 3.0 kV. Repetitive magnetic scanning (60 scans mini’) over the mass range of 3&700 was initiated after a suitable delay from the time of sample injection (usually 300 s). All the data were collected and stored for later analysis. The concentrations of mannitol and glucose were estimated by a comparison of the GC/MS peak areas with the areas produced by known amounts of authentic standards. Glucose uptake. Sporozoites suspended in PBS (lo6 mll’) were incubated at 41’C, for 12 h with 100 PM-glucose enriched with [U-‘4C]glucose (3.7 x 10’ Bq per lo6 sporozoites). During this time sporozoites remained viable and showed no morphological changes. Ten microlitre aliquots were collected every 2 h and washed (5 ml PBS) on Millipore HA 0.22 pm filters. The filters were placed in scintillation vials containing scintillation fluor (OptiPhase ‘HiSafe’ II, LKB) and the radioactivity counted in a LKB-Wallac 1209 Rackbeta liquid scintillation spectrometer. Sporozoites labelled for 6 and 12 h were also treated with 40% trichloroacetic acid (TCA) and radioactivity was counted in precipitated material. Pulse-chase labelling. Sporozoites were resuspended in PBS ( 10bml ‘) and incubated with [U-‘4C]glucose (1.85 x 10’ Bq per lo6 sporozoites) at 41°C for 30 min, centrifuged and washed twice with PBS. They were then resuspended in PBS containing 10 mM of glucose and incubation was continued for a further 90 min. Aliquots of the sporozoite suspension were collected every 30 min and washed twice with PBS. The sporozoites were resuspended in 100 ~1 of distilled water and disrupted by sonication (MSE Soniprep, 3 x 10 s at 70% power output). Radioactively labelled carbohydrates in the sporozoite aliquots were separated and identified by thinlayer chromatography. The 10 ~1 samples (equivalent to 10’ sporozoites) were spotted onto Cellulose F plastic sheets (Merck) and developed with ethyl acetate/acetic acid/formic acid/water (18/3/l/4, v/v). The sheets were then dried in air and developed with n-butanollacetic acid/water (5/l/2, v/v). It is worth noting that only the application of TLC with two developing systems resulted in a satisfactory resolution of mannitol from glucose. Radioactively labelled compounds were located by autoradiography and were identified by comparing their positions on chromatograms with those of individually chromatographed “C-labelled glucose and mannitol. [U-‘4C]Glucose (8.48 GBq mmoll’) and [l“‘C]mannitol(2.18 GBq mmoll’) were obtained from Amersham. The autoradiograms were scanned with a recording densitometer (Isco, Inc., model 1312/UA-5). Peaks were integrated using computer software (Electrophoresis Data Reduction System GS 365) designed by Hoefer Scientific Instruments. Sporozoite viability test. The viability of the sporozoites was measured by the vital dye (fluorescein diacetate, FDA) staining technique described by Jackson, Pappas & Hansen (1985). Sporozoites (10’ mll’) suspended in PBS (pH 7.8) were treated with FDA for 20 min at 28°C. Fluorescein
W. P. MICHALSKI, J. A. EDGAR and S. J. PROWSE
1160 accumulated fluorescence
in living microscope.
cells
was
then
detected
under
a
Protein content assay. Protein content was determined by the bicinchoninic acid (BCA) method (Wiechelman, Braun & Fitzpatrick, 1988) using bovine serum albumin (fraction V) as a standard protein. The estimation of protein content in crude homogenates of oocysts and sporocysts appeared unreliable due to the presence of large quantities of interfering substances, thus it could not be used as a basis of quantitative calculations. RESULTS
Mannitol content in E. tenella Mannitol MS
and
has
been
its content
identified was
during sporulation in E. tenella by GC/
determined
as
the
TMS-
in mannitol extracts of various stages of the parasite development. Kinetic analysis of the mannitol content in oocysts, sporocysts and sporozoites during sporulation is summarized in Fig. 2. Polyol extracts of freshly prepared unsporulated oocysts (frozen in liquid nitrogen immediately after preparation) contained relatively small amounts of mannitol (50-80 nmoles per lo6 oocysts, Fig. 2). During the early stages of sporulation (first &6 h) the amount of detectable mannitol increased approximately six-fold and remained almost unchanged for a further 4 h. A similar pattern of changes in mannitol content was observed during early sporogony in oocysts which had been stored for 24 h at 4°C prior to the mannitol extraction. However, in these oocysts the initial mannitol level was 20&240 nmoles per lo6 oocysts, but increased to approximately 300 nmoles per lo6 oocysts after the first 6 h of sporulation (data derivatives,
ol,“‘.“.“,‘,‘l”“““’ 0
6
12
16
24
Sporulkion
30 time
36
42
46
(hours)
FIG. 2. Changes of mannitol content in oocysts (O), sporocysts ( n ) and sporozoites (A) during sporulation in E. tenellu. Mannitol was identified and its content estimated by the GC/MS method as described in the Material and Methods Section. The values are averages from four experiments f SD. Measurements were made in duplicate in each experiment. The mannitol content is expressed in nmoles per 10600cysts on the basis that one oocyst contains four sporocysts or eight sporozoites.
0
6
12
18 Sporulation
24
30 time
36
42
46
Ihours)
FIG. 3. Changes in contents of amylopectin (m), free glucose (A) and mannitol (0) in sporulating oocysts of E. tenella. The values are averages from three experiments (two measurements in each experiment) f SD. Mannitol and glucose contents in extracts were determined by the GC/MS method. The amylopectin content is expressed in nmoles of glucose obtained by acid hydrolysis of amylopectin granules. In this case the glucose content was determined enzymatically as described in the Material and Methods Section.
not shown). In both types of oocyst preparations the mannitol content slowly diminished during the next 40 h of incubation and was lower than 60 nmoles per IO6 oocysts in sporulated oocysts. Four sporocysts develop within each oocyst during sporulation and can be obtained from oocysts sporulated for at least 12-14 h (Levine, 1982). The mannitol content of sporocyst-enriched fractions prepared from oocysts sporulated for 1248 h was almost identical to that of sporulating oocysts, i.e. it decreased during this time approximately four-fold. These results indicate that all mannitol detected in oocysts sporulated for longer than 12 h had accumulated within sporocysts. Viable sporozoites of E. tenella can be obtained by in vitro excystation from sporocysts prepared from oocysts sporulated for a minimum of 2&24 h. Mannitol was detected in sporozoites obtained from oocysts sporulated for 24 and 36 h but the concentration was relatively low and could only account for 5&60% of that detected in sporocysts. In sporozoites from oocysts sporulated for 48 h the mannitol content was almost identical with that of the oocysts (52 nmoles per 8 x lo6 sporozoites or lo6 oocysts). Very small amounts of mannitol were also detected in merozoites prepared from second stage meronts and varied from 1.2 to 2.0 nmoles per 8 x lo6 merozoites. Merozoite preparations are often contaminated with epithelial cells but no mannitol was detected in these cells.
cycle in E. tenella
Mannitol TABLE l-
1161
Eimeriatenella
ACTIVITIESOFTHEMANN~TOLCYCLEENZYMESINVAR~OUSSTAGESOFDEVELOPMENTOF
Specific activity?
[(units mg protein-‘)
x IO’]
Enzyme* (substrate) Sporulating Oh M-I-PDH (F-6-P/NADP) M-I-Pase (M-l-P) MDH (mannitol) Hexokinase (fructose) (glucose)
23 f 5.3
oocysts
Merozoites
12h
48 h
2Ojz4.8
10f 1.6
12f3.3
115f
65 f 17.8
13f 1.5
47f
15f
130f
310f29.7
Sporozoites
18.7
53f3.8
48f4.1
987 f 146 165Ozk311
1012f 106 1475f241
14.5
55f3.7 690 f 97 876f 103
1.0
445f23 419f 13
ND
1.0
ND ND
* Enzyme and substrate abbreviations and techniques used for measuring enzyme activities are given in the Material and Methods Section. t One unit is expressed in pmoles of substrate per product per min. The values are the average from four estimations f SD. ND, Not determined.
Start
abc
d
e
f
g
FIG. 4. Pulse-chase
labelling of sporozoites with “‘C-glucose. Sporozoites were labelled and carbohydrates analysed by thin-layer chromatography as described in the Material and Methods Section. The photograph represents an autoradiogram of TLC sheet. The analysed samples were: (a) “C-glucose (3.7 x 10’ Bq), (b) “C-glucose and %mannitol, (c) %mannitol (3.7 x 10’ Bq), (d) sporozoites incubated
with “C-glucose for 30 min, (e), (f) and (g) labelled for a further 30,60 and 90 min, respectively.
Amylopectin andfree glucose content Kinetic analysis of the changes in amylopectin and glucose concentration during sporulation, compared with changes in mannitol content, is presented in Fig. 3. Unsporulated oocysts of Eimeria spp. contain large quantities of amylopectin (polyglucose) granules (Wilson & Fairbairn, 1961; Ryley, Bentley, Manners & Stark, 1969; Wang et al., 1975). Acid hydrolysis of granules purified from unsporulated oocysts yielded 468 nmoles of glucose per lo6 oocysts. During early sporogony the amylopectin content decreased markedly, and acid hydrolysis of granules from oocysts sporulated for 8-12 h yielded approximately 130
nmoles glucose per lo6 oocysts. Similar amounts of amylopectin were detected in oocysts sporulated for a further 36 h. In sporozoites excysted in vitro from oocysts sporulated for 48 h the amylopectin content was virtually the same as that of oocysts after 48 h incubation (about 60 nmoles glucose per 8 x lo6 sporozoites). Unsporulated oocysts contained a detectable amount of free glucose as determined by both the enzymic and GC/MS methods. The glucose content diminished rapidly during early sporulation and after 8 h was only detectable by the GC/MS method. Similarly a very small amount of free glucose was detected in sporozoites obtained from oocysts which had been sporulated for 48 h (less than 5 nmoles per 8 x lo6 sporozoites, Fig. 3). Enzymes of mannitol cycle Activities of the mannitol cycle enzymes were monitored in oocysts, sporozoites and merozoites of E. tenella (Table 1). In sporulating oocysts and sporozoites all four key enzymes of the cycle were active. The specific activities of the enzymes fall into two groups. Hexokinase and M- 1-Pase had relatively high activities while M-l-PDH and MDH had much lower specific activities. During early sporogony (12 h sporulation) the activity of M-I-Pase declined by about 60% but remained unchanged during the next 36 h. The activity of MDH was unchanged during sporulation whereas the activities of hexokinase and M- 1-PDH declined markedly during late sporogony. In sporozoites the activities were similar to those detected in oocysts which had been sporulated for 48 h,
1162
W.P. MICHALSKI,J. A. EDGAR and S. J.PROWSE
indicating that these enzymes were probably located within sporozoites. Merozoites obtained from second stage meronts had detectable but very low activities of M-I-Pase and MDH. “C-Glucose uptake by sporozoites Sporozoites incubated in vitro with “C-glucose accumulated the labelled precursor continuously for 12 h at a rate of approximately 5 nmoles per lo6 sporozoites per hour. In sporozoites labelled for 6 or 12 h, approximately 40% of the radioactivity was found to be associated with TCA precipitated (proteinous) material. In order to determine whether glucose served as a substrate to the mannitol cycle, the sporozoites were also pulse-chase labelled with 14Cglucose. Thin layer chromatography, designed to detect labelled mannitol in the presence of glucose, was applied for analysis of radioactive carbohydrates accumulated by sporozoites in these experiments (Fig. 4). After pulse labelling for 30 min sporozoites contained clearly detectable “‘C-glucose as well as some unresolved labelled material at the origin (Fig. 4d). Pulse-chase labelling of sporozoites for a further 90 min revealed that “C-glucose had been converted into a compound which could be identified from its chromatographic behaviour as “C-mannitol (Fig. 4eg). As determined by densitometric analysis of autoradiograms the amount of labelled mannitol increased approximately four-fold during the last 60 min of incubation (Figde-f). These results are in agreement with the other set of data, i.e. the presence of mannitol (Figs. 2 and 3) and the mannitol cycle enzymes (Table 1) in sporozoites indicates that sporozoites have a functional mannitol cycle and can use glucose as a substrate. DISCUSSION Amylopectin, a branched polysaccharide found in almost all stages of the E. tenella life-cycle, has been thought to be the parasite’s only carbohydrate reserve (Ryley et aI., 1969). It has been suggested that amylopectin (effectively glucose) provided the energy needed for the early stages of sporulation, whereas the oxidation of lipids supported the final stages, as well as the metabolism during dormancy (Wilson & Fairbairn, 1961). However, other studies by Vetterhng & Doran (1969), Wang et al. (1975) and Nakai & Ogimoto (1989) seem to support the hypothesis that amylopectin is also the energy source for excystation and the subsequent penetration of cells by sporozoites. Recently Schmatz et aI. (1988, 1989) identified mannit01 as another carbohydrate component of E. tenella oocysts and demonstrated the presence of the mannitol cycle (Fig.1). Studies of the function of the mannitol cycle in E. tenella were limited to the
unsporulated oocysts and it has been postulated that mannitol is generated in the gut during late oogenesis (Schmatz et al., 1988, 1989; Schmatz, 1989). In this paper we present a detailed kinetic analysis of the carbohydrate content in E. tenella oocysts during sporulation and the characterization of the mannitol cycle is extended to the intracellular stages of the parasite. In our experiments, freshly prepared oocysts (frozen immediately after preparation) contained large quantities of amylopectin and a relatively small amount of mannitol (Figs. 2 and 3). During early stages of sporogony the amount of mannitol increased six-fold. This was accompanied by a rapid decrease in amylopectin and free glucose (Fig.3) suggesting that mannitol was synthesized in oocysts from glucose released from amylopectin. This hypothesis appears to be substantiated by the presence of four key enzymes of the mannitol cycle in sporulating oocysts (Schmatz et al., 1989 and Table 1). The activity of amylopectin phosphorylase has also been detected in sporulating oocysts and it has been suggested that this enzyme plays a major role in amylopectin utilization (Wang et al., 1975). All the enzymes of the mannitol cycle are very substrate-specific with the exception of hexokinase, which accepts either fructose or glucose. The pathway is unidirectional since the reaction catalysed by M-1-Pase is irreversible (Schmatz et al., 1988, 1989 and Fig. 1). Unsporulated oocysts have high activities of hexokinase and M-1-Pase, and relatively low activities of MDH. Thus in the presence of a high glucose concentration the activity of the cycle would result in the synthesis of mannitol. In contrast, a dramatic decrease in mannitol content (Figs. 1 and 2) during late sporogony can be explained by declined activities of hexokinase, M-I-PDH and M-1-Pase (Table 1). In addition, high mannitol levels might also inhibit some glycolytic enzymes as described for mycorrhiza (Wedding&Harley, 1976) or down-regulate glycolysis by fructose-6-phosphate withdrawal. These results indicate that mannitol is not only a reserve carbon source but is also metabolically very active and may be simultaneously formed and utilized through the cycle. It has been suggested that the mannitol cycle probably functions as an endogenous energy source as proposed in fungi (Hult & Gatenbeck, 1979). Thus, in sporulating oocysts the activity of the cycle (mannitol synthesis and oxidation) would be dependent on the redox state of the nicotinamide adenine nucleotide system and the reactions would be important in maintaining the correct redox states of these coenzyme pools (Schmatz et al., 1989; Schmatz, 1989). It seems probable that a major metabolic switch takes place in the middle of sporogony when a change from amylopectin to mannitol metabolism occurs. It has previ-
Mannitol cycle in E. tenelia
ously been observed that amylopectin phosphorylase activity steadily declined during oocyst s~~lation (Wang et al., 1975). It appears that the presence of the mannitol cycle is not limited to the oocysts. Key enzymes have been detected in sporozoites and merozoites, indicating that this pathway also operates in the intracellular stages of the parasite’s development (Table 1). This is substantiated by the observation that sporozoites are capable of converting exogenous glucose into mannitol (Fig. 4). There is no evidence in the literature of mannitol being used by the host, which suggests that it acts as an exclusive energy source for the parasite. In addition, the accumulation of mannitol during the intracellular development of the parasite could act as a glucose ‘sink’, allowing the storage of energy with minimal effect on glucose transport through the host and/or parasite membranes facilitating a constant influx of energy (Schmatz, 1989). It has been suggested that mannitol can act as an osmoregulator keeping the oocysts rigid until the end of sporulation. Another possibility is that mannitol is a component of the sporocyst and/or oocyst wall. A polymer of mannitol phosphate known as mannitol techoic acid has been identified in bacteria (Anderton & Wilkinson, 1985). Indeed, the amount of mannitol in sporozoitescould not account for the total mannitol content found in oocysts and sporocysts (from which sporozoites were obtained, Fig. 2). It is also possible that E. tenefla sporozoites are dramatically affected by exposure to superoxide ions and that exogenous mannitol has a protective effect on parasite viability (Michalski & Prowse, 1991). technical assistance of MS Fiona Bodey is gratefully acknowledged. The authors are grateful
Acknowledgements-The
to MS Bernadette Keohane for the GC/MS measurements. The work was supported by a grant from the Chicken Meat Research and Development Council of Australia.
REFERENCE AMES B. N. 1966. Assay of inorganic phosphate, total phosphate and phosphatases. In: Methods in Enzymology, Vol. 8 (Edited by NEUFELD E. F. & GINSBURG V.), pp. 115118. Academic Press, New York. ANDERTON W. 3. & WILKINSON S. G. 1985. Structural studies of a mannitol teichoic acid from the cell wall of bacterium N.C.T.C. 9742. Biochemical Journal 226: 587.-599. HULT K. & GATENBECK S. 1978. Production of NADPH in the mannitol cycle and its relation to polyketide formation in Afternaria alternata. European Journal of Biochemistry88: 607-612. HULT K. & GATENBECK S. 1979. Enzyme activities of the mannitol cycle and some connected pathways in Alrernaria
1163
alternata, with comments on the regulation of the cycle. Acta Chemica Sca~ina~ica 33: 239-243.
JACKSONP. R., PAPPASM. G. & HANSENB. D. 1985. Fluorogenic substrate detection of viable intracellular and extracellular pathogenic protozoa. Science 227: 435-438. LEVINEN. D. 1982. Taxonomy and life cycles of coccidia. In: The Biology ofthe Caccidia (Edited by LONG P. L.), pp. l33. Edward Arnold, London. LEWISD. H. & SMITHD. C. 1967. Sugar alcohols (polyols) in fungi and green plants. I. Distribution, physiology and metabolism. New Phytologtit 66: 143-184. MICHALSKI W. P. % PROWSE S. J. 1991. Superoxide dismutases in Eimeria teneila. Molecular and Biochemical Parasitology 47: 189-195.
NAKAIY. & OGIMOTO K. 1989. Amylopectin as an energy source of Eimeria teneI!a sporozoite infection. In: Coccidiu and Intestinal Coccidiomorphs, Proceedings Vth International Coccidiosis Conference, Tours, France (Edited by IVOREP.), pp. 129-132. Institute National de la Recherche Agronomique, Paris (Colloques de I’INRA, No. 49). PROWSE S. J. & PALLISTER J. 1989. Interferon release as a measure of the T-cell response to coccidial antigens in chickens. Avian Pathology 18: 619-630. RYLEYJ. F., BENTLEY M., MANNERS D.J. & STARKJ. R. 1969. Amylopectin, the storage polysaccharide of the coccidia Eimeria brunetti and E. tenella. Journal of Parasitology 55:
839-845.
SCHMATZ D. M., ARISONB.H., DASHKEVICZ M.P., LIESCH J. M. & TURNER M.J. 1988. Identification and possible role of Dmannitol and 2-0-methyl~hiro-inositol {quebrac~tol) in Eimeriu tenella. Molecular and Biochemical Parasitology 29: 29-36. SCHMATZD. M. 1989. The mannitol cycle - a new metabolic pathway in the coccidia. Parasifology Taday 5: 205-208. SCHMATZ D. M., BACINSKY W. F. & TURNERM. J. 1989.
Evidence for and characterization
of a mannitol cycle in
Eimeria tenella. Molecular and Biochemical Parasitology 32: 263-270. VET~RLING J. M. & KORAN D. J. 1969. Storage polysaccharide in coccidial sporozoites after excystation and penetration of cells. Journal of Protozoology 16: 772-775. WANG C. C., WEPPELMAN R. M. & LOPEZ-RAMOS B. 1975. Isolation of amylopectin granules and identification of amylo~tin phosphoryiase in the oocysts of Eimeria tenelfa. Journal o~Par~~ifology 22: 560-564. WEDDING R. T. & HARLEY J. L. 1976. Fungal polyols metabolites in the control of carbohydrate metabolism of mycorrhizal roots of beech. New Phytaiogist 77: 675-688. WIECHELMANK. J., BRAUN R. D. & FITZPATRICKJ. D. 1988. Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation. Analytical Biochemistry 175: 231-237. WILSON P. A. G. & FAIRBAIRN D. 1961. Biochemistry of spor~ation in oocysts of Eimeria acer~~ina. Journal of Protozooiogy8: 41W16.
MANNITOL
I I57-I 163, I992
METABOLISM
WOJTEK P. MICHALSKI,* CSIRO
Division
of Animal
Health,
Animal
IN EIMERIA
TENELLA
JOHN A. EDGAR and STEPHEN Health
Research
Laboratory,
Parkville,
J. PROWSE Victoria
3052, Australia
(Received 3 February 1992; accepted 19 June 1992)
Ah&act-MICHALsKI W. P., EDGAR J. A. and PROWSES. J. 1992. Mannitol metabolism in Eimeria tenella. International Journalfor Parasitology 22: 1157-l 163. Unsporulated oocysts of Eimeria tenella contain large quantities of carbohydrates, namely amylopectin, mannitol and glucose. Analysis of the carbohydrate content of sporulating oocysts revealed that mannitol content increased markedly during early stages of sporogony (first 4-6 h) but slowly diminished during the next 40 h of sporulation. Accumulation of mannitol was accompanied by a rapid decrease in amylopectin and free glucose, suggesting that mannitol might be synthesized from glucose released from amylopectin. Mannitol was also detected in sporozoite and merozoite extracts. All four mannitol cycle enzymes were detected in oocysts. Sporozoites excysted in vitro had lower activities of all four enzymes. Mannitol-1-phosphatase and mannitol dehydrogenase activity was also detected in merozoites obtained from the second stage schizonts. Sporozoites incubated with ‘%glucose accumulated radioactively labelled precursor continuously for over 12 h and some of the ‘%-glucose was converted into %mannitol. These results indicate that mannitol plays an important role in the metabolism and development of the intracellular stages of the parasite. INDEX KEY sporozoites.
WORDS:
Mannitol;
carbohydrate
metabolism;
INTRODUCTION Coccrorosis is an economically important parasitic disease of poultry which resulted from infection by protozoa of the genus Eimeriu. One of the better characterized species, E. tenella, infects and destroys the epithelial cells lining the caeca resulting in high morbidity and mortality. Chickens are infected by coccidia following ingestion of the vegetative stage of the parasite, the sporulated oocyst. Infective sporozoites are released from oocysts in the intestine and rapidly invade epithelial cells of the caeca. Sporozoites then undergo several generations of asexual reproduction (merogony) before entering a stage of sexual differentiation leading to the formation of oocysts (oogony). Unsporulated oocysts are then shed in the faeces and sporulate when exposed to the appropriate levels of oxygen and humidity (Levine, 1982). Beyond these requirements the oocyst is entirely dependent on endogenous energy to support the process of sporulation. Amylopectin, a branched polysaccharide (polyglucose), is found in granules in almost all stages of the E. tenella life-cycle (Wang, Weppelman & Lopez-Ramos, 1975). Recently, it has been found that oocysts and sporozoites also contain mannitol previ-
Eimeriu
renellu; sporulating
oocysts;
ously reported exclusively in plants and bacteria (Lewis & Smith, 1967; Schmatz, Arison, Dashkevicz, Liesch & Turner, 1988; Schmatz, 1989). Many fungi contain mannitol and various theories have been proposed to explain its role, the most convincing of which is its use in the mannitol cycle (Hult & Gatenbeck, 1978, 1979). A similar mannitol cycle has recently been observed in E. tenella. All four mannitol cycle enzymes namely, mannitol-l-phosphate dehydrogenase (MlPD), mannitol-1-phosphatase (M-lPase), mannitol dehydrogenase (MDH) and hexokinase were found in cell-free extracts of unsporulated oocysts of E. tenella (Schmatz, Baginsky & Turner, 1989 and Fig. 1). It has not been established whether any of the other stages of the parasite possess mannitol cycle enzymes (Schmatz, 1989). At present, information on the function of the mannitol cycle in the coccidia is limited to the unsporulated oocysts and it has been postulated that mannitol is generated by the parasite in the gut during late oogenesis (Schmatz et al., 1988). In this paper we present evidence indicating that mannitol is also synthesized in sporulating oocysts and sporozoites from glucose. MATERIAL AND METHODS
* To whom all correspondence
should be addressed.
Oocystpreparation andsporulation. A line of E. tenella was established in our laboratory from a single oocyst obtained 1157
1158
W. P. MICHALSKI. J. A. EDGAR and S. J. PROWSE
Mannitol
Cycle
Glucose-&phosphate
QlUCO8~-6-P imMner~*e
It II
Fructose-&phosphate
Mannitol-l-phosphate
Fructose
Mannitol
FIG. 1. The mannitol cycle in Eimeria tenella. The main enzymes involved are: mannitol- l-phosphate dehydrogenase (M-I-PDH), mannitol-1-phosphatase (M-I-Pase), mannitol dehydrogenase (MDH) and hexokinase. Glucose is converted to fructose-dphosphate via hexokinase and glucose-6-phosphate isomerase. Fructose-6-phosphate can enter the mannitol cycle via M-I-PDH and is reduced to mannitol-l-phosphate which in turn is dephosphorylated by M-1-Pase to yield mannitol. Mannitol is then oxidized to fructose by MDH and is rephosphorylated to fructose-6phosphate by hexokinase. Note that M-I-PDH can also oxidase NADPH (Schmatz et al., 1989).
from a field sample. The line was passaged in SPF birds aged between 3 and 6 weeks. Oocysts were prepared from the caeca of infected birds as described by Prowse & Pallister (1989) except that mucosal homogenate was digested with trypsin at 41°C for only 2 h. Freshly prepared oocysts were resuspended in sterile phosphate buffer-saline (PBS) and immediately sparged with high purity nitrogen for 15 min and kept under anaerobic conditions at 4’C until needed. For the kinetic experiments they were used immediately. Sporulation was achieved by incubating 100 ml of the oocyst suspension (5 x lo6 ml-‘) in a 500 ml flask in a 28°C waterbath at 100 beats min-‘. Samples of sporulating oocysts (3 x 10’) were collected every 2 h under sterile conditions for mannitol extraction. They were then sparged with nitrogen and snapfrozen in liquid nitrogen. Isolation of sporocysts. Fractions enriched in sporocysts were obtained as follows: oocysts sporulated for 12, 24, 36 and 48 h were fractured by mixing oocysts with glass beads on
a vortex mixer. Mixing was continued (approximately 8-10 min) until no oocysts were detected under the microscope. Homogenates containing mostly sporocysts and cell debris were washed twice in PBS and examined under the microscope. These fractions enriched in sporocysts contained no oocysts or sporozoites and were used immediately for mannitol extraction. Isolation of sporozoites and merozoiies. Sporozoites were obtained from oocysts sporulated for 24, 36 and 48 h using the method described previously (Prowse & Pallister, 1989). The sterile sporozoite preparations were either used immediately in enzyme activity assay or pelleted and snapfrozen in liquid nitrogen for mannitol extraction. For merozoite isolation, chickens were inoculated with 0.5-l .O x 10’ oocysts and the caeca removed 113 h later. They were slit open and washed in cold PBS until clean. The mucosa was scraped off and homogenized in a Waring blendor for 20 s in PBS containing 2 mg ml-’ glucose. The homogenate was transferred to a tube and the clumps allowed to settle. The supernatant was filtered through eight layers of gauze and centrifuged at 15OOxg for 10 min. The pellet was then resuspended in 50 ml of cold lysis buffer containing 8.3 g I-‘NH,Cl,0.372 g I-‘EDTA,l g 1.‘K&O, pH 7.6. After 30 min on ice the suspension was diluted in 2 vol. of PBS/glucose and centrifuged at 1500 x g for 10 mm and suspended in 30 ml of PBS/glucose. The suspension was then passed down a nylon wool column containing 1.6 g of washed nylon wool in a 10 ml syringe. The merozoites were washed off the column with PBS, centrifuged as above and counted. On average the merozoite preparations contained approximately IO-15% epithelial cells. Epithelial cells were obtained from the mucosa of uninfected chickens in the same manner as described for merozoite preparation, except that the cells were not passed down a nylon wool column. Preparation of crude enzyme. Crude enzyme homogenates were obtained from unsporulated (10’) and sporulated (5 x 10’) oocysts using the method described previously (Schmatz et al., 1989). Crude enzyme preparations from sporozoites (10’) and merozoites (108)were also obtained using the same method except that the cells were homogenized by sonication (MSE Soniprep, 3 x 10 s at 70% power output) at 4°C. Crude homogenates from epithelial cells of uninfected chickens were also prepared by sonication. All homogenates were desalted on a Bio-Gel P-6DG column before the final concentration step. Oocyst homogenates contained approximately 8-10 mg protein ml-‘, whereas the preparations from sporozoites and merozoites contained 2-3 mg protein ml ’ Assays for enzyme activities. All enzyme assays were initiated with substrate and monitored for 8 min unless stated otherwise. Determinations were made in 1 ml cuvettes by continuous scanning against appropriate reaction blanks. Dehydrogenase activities were determined by measuring coenzyme reduction or oxidation spectrophotometricaily at 340 nm in a Hitachi U-2000 spectrophotometer. Specific activity of these enzymes is expressed in pmoles of nicotinamide adenine nucleotides reduced or oxidized per min (one unit) per mg of protein. The activity of mannitol-lphosphatase is expressed in pmoles of phosphate released per min (one unit) per mg of protein. Manniiol- 1-phosphate dehydrogenase (M- I-PDH, EC
Mannitol
cycle in E. tenella
1.1.1.17). The enzyme activity was measured at 25°C in a reaction mixture containing 50 mM-HEPES @H 7.5), 1 mMEDTA, 1 mM-NAD (P)H, 2.5 mM-fructose-6-phosphate (F6-P) and the crude enzyme preparation. The reverse reaction was determined at 25°C in a reaction mixture containing 50 mM-3-( 1, I-dimethyl-2-hydroxyethyl)-2-hydroxypropane sulphonic acid (AMPSO; pH 9.5), 1 mM-EDTA, 2 mM-NADP, 50 mM-mannitol-l-phosphate (M-l-P) and the crude enzyme. Mannitol-I-phosphatase (M-I-Pase, EC 3.1.3.22). The enzyme was assayed at 25°C in 50 mM-HEPES (pH 7.0) containing 1 mM-mannitol-l-phosphate, 1 mM-EDTA and crude enzyme. Free phosphate released during the 15 min incubation was determined using the Ames reagent (Ames, 1966). Mannirol dehydrogenase (MDH, EC 1.1.1.67). MDH activity was determined at 25°C in a reaction mixture containing 50 mM-AMPS0 (pH 9.0), 1 mM-EDTA, 3.0 mMNAD, 50 mM-mannitol and the crude enzyme preparation. Hexokinase (EC 2.7.1.1). Hexokinase was assayed at 25°C in a coupled reaction system as described by Schmatz et al. (1989). The reaction mixture contained 50 mM-HEPES (pH 7.5) 5 mM-MgCl,, 2 mh+ATP, 0.25 mht-NADP, 5 mMfructose, glucose-6-phosphate dehydrogenase (10 units, Sigma G-5885), phosphoglucose isomerase (5 units, Sigma P5538) and the crude enzyme. In some assays, fructose was substituted by glucose to determine substrate specificity. Amylopectin and free glucose determination. Amylopectin granules were prepared from oocysts (2 x 106) according to the method of Wang er al. (1975). Amylopectin content was determined as the amount of glucose released after acid hydrolysis. Amylopectin samples were hydrolysed in glass ampules (sealed under nitrogen) with 0.6 N-HCl at 105’C for 2.5 h. Hydrolysates were then neutralized with 0.5 i+NaOH and the glucose concentration determined by the enzymatic method (glucose oxidase/peroxidase test kits, Sigma Diagnostics or Boehringer Mannheim Diagnostica) using a freshly prepared glucose solution as an external standard. Free glucose in supernatants of crushed oocysts was measured by the enzymatic method. Gas chromatography/mass spectrometry (GC/MS) analysis was used for glucose determination in mannitol extracts (see below). Mannitol extraction. Preparations of oocysts and sporocysts (both 3 x 10’) were resuspended in 1 ml of double distilled water and homogenized with glass beads. Sporozoites (3 x lo*), merozoites (5 x 10’) and epithelial cells (10”) were resuspended in double distilled water and homogenized by sonication (MSE Soniprep, 3 x 10 s at 70% power output). Mannitol extracts were prepared from the homogenates using the procedure described previously (Schmatz et al., 1988). Preparation of trimethylsilyl derivatives and chromarography. Extracted fractions and standards were derivatized by trimethylsilization (TMS). Dried extracts were dissolved in equal volumes of anhydrous pyridine and bis(trimethylsilyl)trifluoroacetamide (BSTFA, Sigma), and incubated for 12 h at 60°C. These TMS-derivatized samples were injected directly into the gas chromatography system. TMS-derivatized samples were analysed by GC/MS. GC/MS was carried out on a JOEL JMS-DX 303 mass spectrometer coupled to a Varian Vista 6000 gas chromatograph and a
1159
JOEL JMA-DA 5000 data system. The vitreous silica capillary column (25 m x 0.33mm ID) with BP1 (SGE) as the liquid phase (0.5 pm film) was connected directly to the ion source and temperature programmed from 180 to 280°C at either 10 or 4°C mini’. Electron impact mass spectra were generated using electrons at 70 eV and an accelerating voltage of 3.0 kV. Repetitive magnetic scanning (60 scans mini’) over the mass range of 3&700 was initiated after a suitable delay from the time of sample injection (usually 300 s). All the data were collected and stored for later analysis. The concentrations of mannitol and glucose were estimated by a comparison of the GC/MS peak areas with the areas produced by known amounts of authentic standards. Glucose uptake. Sporozoites suspended in PBS (lo6 mll’) were incubated at 41’C, for 12 h with 100 PM-glucose enriched with [U-‘4C]glucose (3.7 x 10’ Bq per lo6 sporozoites). During this time sporozoites remained viable and showed no morphological changes. Ten microlitre aliquots were collected every 2 h and washed (5 ml PBS) on Millipore HA 0.22 pm filters. The filters were placed in scintillation vials containing scintillation fluor (OptiPhase ‘HiSafe’ II, LKB) and the radioactivity counted in a LKB-Wallac 1209 Rackbeta liquid scintillation spectrometer. Sporozoites labelled for 6 and 12 h were also treated with 40% trichloroacetic acid (TCA) and radioactivity was counted in precipitated material. Pulse-chase labelling. Sporozoites were resuspended in PBS ( 10bml ‘) and incubated with [U-‘4C]glucose (1.85 x 10’ Bq per lo6 sporozoites) at 41°C for 30 min, centrifuged and washed twice with PBS. They were then resuspended in PBS containing 10 mM of glucose and incubation was continued for a further 90 min. Aliquots of the sporozoite suspension were collected every 30 min and washed twice with PBS. The sporozoites were resuspended in 100 ~1 of distilled water and disrupted by sonication (MSE Soniprep, 3 x 10 s at 70% power output). Radioactively labelled carbohydrates in the sporozoite aliquots were separated and identified by thinlayer chromatography. The 10 ~1 samples (equivalent to 10’ sporozoites) were spotted onto Cellulose F plastic sheets (Merck) and developed with ethyl acetate/acetic acid/formic acid/water (18/3/l/4, v/v). The sheets were then dried in air and developed with n-butanollacetic acid/water (5/l/2, v/v). It is worth noting that only the application of TLC with two developing systems resulted in a satisfactory resolution of mannitol from glucose. Radioactively labelled compounds were located by autoradiography and were identified by comparing their positions on chromatograms with those of individually chromatographed “C-labelled glucose and mannitol. [U-‘4C]Glucose (8.48 GBq mmoll’) and [l“‘C]mannitol(2.18 GBq mmoll’) were obtained from Amersham. The autoradiograms were scanned with a recording densitometer (Isco, Inc., model 1312/UA-5). Peaks were integrated using computer software (Electrophoresis Data Reduction System GS 365) designed by Hoefer Scientific Instruments. Sporozoite viability test. The viability of the sporozoites was measured by the vital dye (fluorescein diacetate, FDA) staining technique described by Jackson, Pappas & Hansen (1985). Sporozoites (10’ mll’) suspended in PBS (pH 7.8) were treated with FDA for 20 min at 28°C. Fluorescein
W. P. MICHALSKI, J. A. EDGAR and S. J. PROWSE
1160 accumulated fluorescence
in living microscope.
cells
was
then
detected
under
a
Protein content assay. Protein content was determined by the bicinchoninic acid (BCA) method (Wiechelman, Braun & Fitzpatrick, 1988) using bovine serum albumin (fraction V) as a standard protein. The estimation of protein content in crude homogenates of oocysts and sporocysts appeared unreliable due to the presence of large quantities of interfering substances, thus it could not be used as a basis of quantitative calculations. RESULTS
Mannitol content in E. tenella Mannitol MS
and
has
been
its content
identified was
during sporulation in E. tenella by GC/
determined
as
the
TMS-
in mannitol extracts of various stages of the parasite development. Kinetic analysis of the mannitol content in oocysts, sporocysts and sporozoites during sporulation is summarized in Fig. 2. Polyol extracts of freshly prepared unsporulated oocysts (frozen in liquid nitrogen immediately after preparation) contained relatively small amounts of mannitol (50-80 nmoles per lo6 oocysts, Fig. 2). During the early stages of sporulation (first &6 h) the amount of detectable mannitol increased approximately six-fold and remained almost unchanged for a further 4 h. A similar pattern of changes in mannitol content was observed during early sporogony in oocysts which had been stored for 24 h at 4°C prior to the mannitol extraction. However, in these oocysts the initial mannitol level was 20&240 nmoles per lo6 oocysts, but increased to approximately 300 nmoles per lo6 oocysts after the first 6 h of sporulation (data derivatives,
ol,“‘.“.“,‘,‘l”“““’ 0
6
12
16
24
Sporulkion
30 time
36
42
46
(hours)
FIG. 2. Changes of mannitol content in oocysts (O), sporocysts ( n ) and sporozoites (A) during sporulation in E. tenellu. Mannitol was identified and its content estimated by the GC/MS method as described in the Material and Methods Section. The values are averages from four experiments f SD. Measurements were made in duplicate in each experiment. The mannitol content is expressed in nmoles per 10600cysts on the basis that one oocyst contains four sporocysts or eight sporozoites.
0
6
12
18 Sporulation
24
30 time
36
42
46
Ihours)
FIG. 3. Changes in contents of amylopectin (m), free glucose (A) and mannitol (0) in sporulating oocysts of E. tenella. The values are averages from three experiments (two measurements in each experiment) f SD. Mannitol and glucose contents in extracts were determined by the GC/MS method. The amylopectin content is expressed in nmoles of glucose obtained by acid hydrolysis of amylopectin granules. In this case the glucose content was determined enzymatically as described in the Material and Methods Section.
not shown). In both types of oocyst preparations the mannitol content slowly diminished during the next 40 h of incubation and was lower than 60 nmoles per IO6 oocysts in sporulated oocysts. Four sporocysts develop within each oocyst during sporulation and can be obtained from oocysts sporulated for at least 12-14 h (Levine, 1982). The mannitol content of sporocyst-enriched fractions prepared from oocysts sporulated for 1248 h was almost identical to that of sporulating oocysts, i.e. it decreased during this time approximately four-fold. These results indicate that all mannitol detected in oocysts sporulated for longer than 12 h had accumulated within sporocysts. Viable sporozoites of E. tenella can be obtained by in vitro excystation from sporocysts prepared from oocysts sporulated for a minimum of 2&24 h. Mannitol was detected in sporozoites obtained from oocysts sporulated for 24 and 36 h but the concentration was relatively low and could only account for 5&60% of that detected in sporocysts. In sporozoites from oocysts sporulated for 48 h the mannitol content was almost identical with that of the oocysts (52 nmoles per 8 x lo6 sporozoites or lo6 oocysts). Very small amounts of mannitol were also detected in merozoites prepared from second stage meronts and varied from 1.2 to 2.0 nmoles per 8 x lo6 merozoites. Merozoite preparations are often contaminated with epithelial cells but no mannitol was detected in these cells.
cycle in E. tenella
Mannitol TABLE l-
1161
Eimeriatenella
ACTIVITIESOFTHEMANN~TOLCYCLEENZYMESINVAR~OUSSTAGESOFDEVELOPMENTOF
Specific activity?
[(units mg protein-‘)
x IO’]
Enzyme* (substrate) Sporulating Oh M-I-PDH (F-6-P/NADP) M-I-Pase (M-l-P) MDH (mannitol) Hexokinase (fructose) (glucose)
23 f 5.3
oocysts
Merozoites
12h
48 h
2Ojz4.8
10f 1.6
12f3.3
115f
65 f 17.8
13f 1.5
47f
15f
130f
310f29.7
Sporozoites
18.7
53f3.8
48f4.1
987 f 146 165Ozk311
1012f 106 1475f241
14.5
55f3.7 690 f 97 876f 103
1.0
445f23 419f 13
ND
1.0
ND ND
* Enzyme and substrate abbreviations and techniques used for measuring enzyme activities are given in the Material and Methods Section. t One unit is expressed in pmoles of substrate per product per min. The values are the average from four estimations f SD. ND, Not determined.
Start
abc
d
e
f
g
FIG. 4. Pulse-chase
labelling of sporozoites with “‘C-glucose. Sporozoites were labelled and carbohydrates analysed by thin-layer chromatography as described in the Material and Methods Section. The photograph represents an autoradiogram of TLC sheet. The analysed samples were: (a) “C-glucose (3.7 x 10’ Bq), (b) “C-glucose and %mannitol, (c) %mannitol (3.7 x 10’ Bq), (d) sporozoites incubated
with “C-glucose for 30 min, (e), (f) and (g) labelled for a further 30,60 and 90 min, respectively.
Amylopectin andfree glucose content Kinetic analysis of the changes in amylopectin and glucose concentration during sporulation, compared with changes in mannitol content, is presented in Fig. 3. Unsporulated oocysts of Eimeria spp. contain large quantities of amylopectin (polyglucose) granules (Wilson & Fairbairn, 1961; Ryley, Bentley, Manners & Stark, 1969; Wang et al., 1975). Acid hydrolysis of granules purified from unsporulated oocysts yielded 468 nmoles of glucose per lo6 oocysts. During early sporogony the amylopectin content decreased markedly, and acid hydrolysis of granules from oocysts sporulated for 8-12 h yielded approximately 130
nmoles glucose per lo6 oocysts. Similar amounts of amylopectin were detected in oocysts sporulated for a further 36 h. In sporozoites excysted in vitro from oocysts sporulated for 48 h the amylopectin content was virtually the same as that of oocysts after 48 h incubation (about 60 nmoles glucose per 8 x lo6 sporozoites). Unsporulated oocysts contained a detectable amount of free glucose as determined by both the enzymic and GC/MS methods. The glucose content diminished rapidly during early sporulation and after 8 h was only detectable by the GC/MS method. Similarly a very small amount of free glucose was detected in sporozoites obtained from oocysts which had been sporulated for 48 h (less than 5 nmoles per 8 x lo6 sporozoites, Fig. 3). Enzymes of mannitol cycle Activities of the mannitol cycle enzymes were monitored in oocysts, sporozoites and merozoites of E. tenella (Table 1). In sporulating oocysts and sporozoites all four key enzymes of the cycle were active. The specific activities of the enzymes fall into two groups. Hexokinase and M- 1-Pase had relatively high activities while M-l-PDH and MDH had much lower specific activities. During early sporogony (12 h sporulation) the activity of M-I-Pase declined by about 60% but remained unchanged during the next 36 h. The activity of MDH was unchanged during sporulation whereas the activities of hexokinase and M- 1-PDH declined markedly during late sporogony. In sporozoites the activities were similar to those detected in oocysts which had been sporulated for 48 h,
1162
W.P. MICHALSKI,J. A. EDGAR and S. J.PROWSE
indicating that these enzymes were probably located within sporozoites. Merozoites obtained from second stage meronts had detectable but very low activities of M-I-Pase and MDH. “C-Glucose uptake by sporozoites Sporozoites incubated in vitro with “C-glucose accumulated the labelled precursor continuously for 12 h at a rate of approximately 5 nmoles per lo6 sporozoites per hour. In sporozoites labelled for 6 or 12 h, approximately 40% of the radioactivity was found to be associated with TCA precipitated (proteinous) material. In order to determine whether glucose served as a substrate to the mannitol cycle, the sporozoites were also pulse-chase labelled with 14Cglucose. Thin layer chromatography, designed to detect labelled mannitol in the presence of glucose, was applied for analysis of radioactive carbohydrates accumulated by sporozoites in these experiments (Fig. 4). After pulse labelling for 30 min sporozoites contained clearly detectable “‘C-glucose as well as some unresolved labelled material at the origin (Fig. 4d). Pulse-chase labelling of sporozoites for a further 90 min revealed that “C-glucose had been converted into a compound which could be identified from its chromatographic behaviour as “C-mannitol (Fig. 4eg). As determined by densitometric analysis of autoradiograms the amount of labelled mannitol increased approximately four-fold during the last 60 min of incubation (Figde-f). These results are in agreement with the other set of data, i.e. the presence of mannitol (Figs. 2 and 3) and the mannitol cycle enzymes (Table 1) in sporozoites indicates that sporozoites have a functional mannitol cycle and can use glucose as a substrate. DISCUSSION Amylopectin, a branched polysaccharide found in almost all stages of the E. tenella life-cycle, has been thought to be the parasite’s only carbohydrate reserve (Ryley et aI., 1969). It has been suggested that amylopectin (effectively glucose) provided the energy needed for the early stages of sporulation, whereas the oxidation of lipids supported the final stages, as well as the metabolism during dormancy (Wilson & Fairbairn, 1961). However, other studies by Vetterhng & Doran (1969), Wang et al. (1975) and Nakai & Ogimoto (1989) seem to support the hypothesis that amylopectin is also the energy source for excystation and the subsequent penetration of cells by sporozoites. Recently Schmatz et aI. (1988, 1989) identified mannit01 as another carbohydrate component of E. tenella oocysts and demonstrated the presence of the mannitol cycle (Fig.1). Studies of the function of the mannitol cycle in E. tenella were limited to the
unsporulated oocysts and it has been postulated that mannitol is generated in the gut during late oogenesis (Schmatz et al., 1988, 1989; Schmatz, 1989). In this paper we present a detailed kinetic analysis of the carbohydrate content in E. tenella oocysts during sporulation and the characterization of the mannitol cycle is extended to the intracellular stages of the parasite. In our experiments, freshly prepared oocysts (frozen immediately after preparation) contained large quantities of amylopectin and a relatively small amount of mannitol (Figs. 2 and 3). During early stages of sporogony the amount of mannitol increased six-fold. This was accompanied by a rapid decrease in amylopectin and free glucose (Fig.3) suggesting that mannitol was synthesized in oocysts from glucose released from amylopectin. This hypothesis appears to be substantiated by the presence of four key enzymes of the mannitol cycle in sporulating oocysts (Schmatz et al., 1989 and Table 1). The activity of amylopectin phosphorylase has also been detected in sporulating oocysts and it has been suggested that this enzyme plays a major role in amylopectin utilization (Wang et al., 1975). All the enzymes of the mannitol cycle are very substrate-specific with the exception of hexokinase, which accepts either fructose or glucose. The pathway is unidirectional since the reaction catalysed by M-1-Pase is irreversible (Schmatz et al., 1988, 1989 and Fig. 1). Unsporulated oocysts have high activities of hexokinase and M-1-Pase, and relatively low activities of MDH. Thus in the presence of a high glucose concentration the activity of the cycle would result in the synthesis of mannitol. In contrast, a dramatic decrease in mannitol content (Figs. 1 and 2) during late sporogony can be explained by declined activities of hexokinase, M-I-PDH and M-1-Pase (Table 1). In addition, high mannitol levels might also inhibit some glycolytic enzymes as described for mycorrhiza (Wedding&Harley, 1976) or down-regulate glycolysis by fructose-6-phosphate withdrawal. These results indicate that mannitol is not only a reserve carbon source but is also metabolically very active and may be simultaneously formed and utilized through the cycle. It has been suggested that the mannitol cycle probably functions as an endogenous energy source as proposed in fungi (Hult & Gatenbeck, 1979). Thus, in sporulating oocysts the activity of the cycle (mannitol synthesis and oxidation) would be dependent on the redox state of the nicotinamide adenine nucleotide system and the reactions would be important in maintaining the correct redox states of these coenzyme pools (Schmatz et al., 1989; Schmatz, 1989). It seems probable that a major metabolic switch takes place in the middle of sporogony when a change from amylopectin to mannitol metabolism occurs. It has previ-
Mannitol cycle in E. tenelia
ously been observed that amylopectin phosphorylase activity steadily declined during oocyst s~~lation (Wang et al., 1975). It appears that the presence of the mannitol cycle is not limited to the oocysts. Key enzymes have been detected in sporozoites and merozoites, indicating that this pathway also operates in the intracellular stages of the parasite’s development (Table 1). This is substantiated by the observation that sporozoites are capable of converting exogenous glucose into mannitol (Fig. 4). There is no evidence in the literature of mannitol being used by the host, which suggests that it acts as an exclusive energy source for the parasite. In addition, the accumulation of mannitol during the intracellular development of the parasite could act as a glucose ‘sink’, allowing the storage of energy with minimal effect on glucose transport through the host and/or parasite membranes facilitating a constant influx of energy (Schmatz, 1989). It has been suggested that mannitol can act as an osmoregulator keeping the oocysts rigid until the end of sporulation. Another possibility is that mannitol is a component of the sporocyst and/or oocyst wall. A polymer of mannitol phosphate known as mannitol techoic acid has been identified in bacteria (Anderton & Wilkinson, 1985). Indeed, the amount of mannitol in sporozoitescould not account for the total mannitol content found in oocysts and sporocysts (from which sporozoites were obtained, Fig. 2). It is also possible that E. tenefla sporozoites are dramatically affected by exposure to superoxide ions and that exogenous mannitol has a protective effect on parasite viability (Michalski & Prowse, 1991). technical assistance of MS Fiona Bodey is gratefully acknowledged. The authors are grateful
Acknowledgements-The
to MS Bernadette Keohane for the GC/MS measurements. The work was supported by a grant from the Chicken Meat Research and Development Council of Australia.
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