1 Haskins Laboratories and Department of Biology, Pace University, New York, NY 10038, USA
2 Department of Chemistry, Pace University, New York, NY 10038, USA
3 Departments of Medicine and Pathology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
4 SLIL Biomedical Corp., Madison, WI 53711, USA
5 Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48202, USA
Correspondence
Cyrus J. Bacchi
cbacchi{at}pace.edu
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ABSTRACT |
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This article is dedicated to the memory of Dr Seymour H. Hutner (31 October 19111 June 2003), mentor, colleague and friend. His dedication to and development of the field of protozoology was an inspiration to all of us.
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INTRODUCTION |
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The microsporidia have become increasingly important as human pathogens in AIDS. There are approximately eight genera with species proven to be pathogenic in humans. Enterocytozoon (Ent.) bienusi, an enteric parasite which causes intractable diarrhoea in AIDS, is the most common species (Wittner & Weiss, 1999; Weber et al., 1994
).
Chemotherapy for microsporidia infections has relied on the benzimidazoles and fumagillin, which are generally effective with exceptions: albendazole is not effective versus Ent. bienusi and fumagillin has cytotoxic effects, especially in AIDS patients (Didier, 1997; Costa & Weiss, 2000
; Dieterich et al., 1994
; Weiss et al., 1994a
; Katiyar et al., 1994
).
With this as background, our work has focused on polyamine metabolism in the microsporidia using Enc. cuniculi as model organism (Bacchi et al., 2001). Polyamines are low-molecular-mass polycations, found universally in cells, which function as cofactors for macromolecule synthesis, in osmotic control, and as structural facilitators for nucleic acids (Cohen, 1998
). The major polyamines are putrescine, spermidine and spermine. Polyamines are absolutely required for cell division and differentiation and their internal concentrations are carefully controlled by synthesis, degradation, excretion and uptake from the environment. In mammals and many protozoa, synthesis occurs from the conversion of ornithine to putrescine through ornithine decarboxylase (ODC) (Bacchi & Yarlett, 2002
), although the enteric parasite Cryptosporidium parvum has a plant-like pathway in which arginine is the starting point for putrescine synthesis through arginine decarboxylase (Keithly et al., 1997
). One or two aminopropyl groups, originating from decarboxylated S-adenosylmethionine, are added to putrescine to form spermidine and spermine, respectively. The limiting enzymes of polyamine synthesis are ODC and S-adenosylmethionine decarboxylase (AdoMetdc), both of which are highly inducible and have short half-lives in the cell (Marton & Pegg, 1995
). Mammalian cells, as well as some protozoa, take up spermine and spermidine, interconverting them to spermidine and putrescine, respectively. The key enzymes in interconversion are spermidine/spermine N1-acetyltransferase (SSAT) and polyamine oxidase (PAO). SSAT is also highly inducible by exogenous polyamines, and the trio of ODC, AdoMetdc and SSAT serves to keep intracellular polyamines at relatively stable levels (Marton & Pegg, 1995
).
Chemotherapeutic interest in the polyamine pathway was initially spurred by the development of enzyme-activated inhibitors of ODC [DL--difluoromethylornithine hydrochloride (DFMO)] and AdoMetdc (MDL 73811, AbeAdo; Bacchi & Yarlett, 2002
). Although these were not effective clinically as anti-tumour agents, DFMO found use clinically against African sleeping sickness (Schechter & Sjoerdsma, 1989
) and AbeAdo in experimental trypanosome infections (Bitonti et al., 1990
). More recent attention, however, has focused on polyamine analogues such as bis(ethyl)norspermine (BE 3-3-3) and bis(ethyl)homospermine (BE 4-4-4) as anti-tumour agents. These agents are taken into mammalian cells on polyamine transporters and down-regulate ODC and AdoMetdc, while up-regulating SSAT. The net result is that polyamine synthesis is shut down while remaining intracellular polyamines are acetylated and excreted. The resulting decline in polyamine levels causes apoptosis and cell death, since the analogues do not substitute functionally for natural polyamines (Marton & Pegg, 1995
; Casero & Woster, 2001
; Frydman & Valasinas, 1999
; Bernacki et al., 1995
).
Our recent work has demonstrated that several classes of polyamine analogues block in vitro growth of Enc. cuniculi and sterilize host monolayers at micromolar levels. These agents also cured well-validated laboratory model infections of Enc. cuniculi (Zou et al., 2001; Bacchi et al., 2002
). In the present study, we examine these analogues for effects on polyamine metabolism in Enc. cuniculi.
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METHODS |
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Enzyme preparations.
Intact mature spores were obtained from supernatant culture media and stored aseptically at 04 °C (Bacchi et al., 2001). Spores were washed in 150 mM PBS [0·9 % (w/v) NaCl] containing 1 % (w/v) SDS and resuspended in PBS prior to breaking. Spores were poured into 2 ml microfuge tubes containing 425600 nm glass beads (Sigma) and disrupted (68 min at 4 °C) using a Mini Bead Beater (Bio Spec Products) or Vortex Mixer and adapter (#13000-50-V1; Mo Bio Laboratories) (Weiss et al., 1994b
). Homogenate preparations with greater than 90 % breakage (by microscopy) were collected and cleared by centrifugation at 2200 g for 10 min. The cleared supernatants were used to determine SSAT and PAO activity.
Gradient purification of pre-emergent spores.
The large-scale gradient procedure described previously (Bacchi et al., 2001) was used to obtain purified preparations of pre-emergent spores. Usually 10 heavily infected (5080 %) monolayers from Falcon T75 flasks were trypsinized, harvested and ground in a glass vessel for 68 min (34x2 min grinding) at 04 °C. The breakage medium contained W-T medium (described below under Incubation studies using pre-emergent spores) plus a protease inhibitor cocktail containing 2·7 mM N
-p-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK), 0·15 mM aprotinin, 2·2 mM leupeptin and 0·5 mM DTT in 50 mM KCl/0·15 M Na3PO4, pH 7·4. Cell homogenates were passed through 12 µm and 5 µm filters and the final filtrate was centrifuged for 10 min at 15 000 g on 50 % (v/v) stock isotonic Percoll (1 ml/11 ml Beckman 14x89 mm tube; Green et al., 1999
). Two bands were obtained, a broad one at 1·0181·035 g ml1 (light band) and a narrower one at 1·1021·119 g ml1 (heavy band). We had demonstrated from the previous study that the heavy band contained pre-emergent spores and immature spores while the light band contained debris, spore cases and some immature stages (Bacchi et al., 2001
). Since the heavy fraction exhibited far greater metabolic activity than the light fraction (Bacchi et al., 2001
), we used it for the data presented in the present study. Cell counts of suspensions in several experiments prior to and post-incubation indicated that no lysis of these forms had occurred.
Polyamine analysis.
Polyamine content was quantified by reversed-phase HPLC using a Perkin-Elmer LC-410 pump and a percosphere C18-5 µ 150x4·6 mm column (Perkin Elmer). Heavy gradient fractions (pre-emergent spores) were extracted with 10 % (w/v) trichloroacetic acid (TCA) at 04 °C. Denatured protein was removed by centrifugation and extracts were pre-column derivatized with 0·8 g o-phthalaldehyde l1 (2 : 1) dissolved in 3 ml methanol added to 30·9 g boric acid l1 containing 24 g KOH l1 (pH 10·4). The derivatized samples were quantified with a Perkin-Elmer LC 240 fluorescence detector using an excitation wavelength of 320 nm and an absorption wavelength of 455 nm. Signals were integrated using -RAM (IN/US Systems) Version 3.1 software. Concentrations of polyamines were determined relative to standards. The HPLC solvent gradient used and other details have been described previously (Yarlett et al., 1994
).
Enzyme assays.
All incubations were at 37 °C. SSAT was measured according to Libby et al. (1989) in a reaction containing 0·06 M Bicine buffer (pH 8·0), 0·156 mM spermine or spermidine, 0·1 µCi (0·5 nmol) [1-14C]acetyl-CoA and 50 µl spore enzyme preparation. Reactions were incubated for 30 min, stopped by addition of 20 µl 0·5 M hydroxylamine, and placed on ice. Reaction tubes were heated for 3 min in a boiling water bath and centrifuged to remove protein. Clarified reactions were spotted on P-81 phosphocellulose filters. Filters were washed with distilled water, then methanol, dried and counted. Activity is expressed as nmol (mg protein)1 (30 min)1.
PAO was measured spectrophotometrically (Beckman DU 640) according to the method of Childs & Bardsley (1975) in 0·1 M glycine buffer (pH 7·0) containing 10 units of horseradish peroxidase [Sigma Type VIA: 1 unit will oxidize 1 µmol 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) min1 at 25 °C at pH 5·0], 5 mM ABTS and 22220 µg Enc. cuniculi protein. Final volume was 1 ml. Assays (in duplicate) were started by addition of 1 mM N1-acetylspermine. Assays were linked to reduction of ABTS by peroxidase and measurement of oxidized product at 420 nm, using a six-compartment cell holder at 37 °C. The extinction coefficient of ABTS at 420 nm is E=3·6x104 M1 cm1 (Childs & Bardsley, 1975
). Studies examining polyamine analogues as substrates of PAO had N1-acetylspermine left out of the reaction. Blanks containing protein but no N1-acetylspermine were run with each reaction, and subtracted from substrate runs. Initial velocity plots were obtained using Beckman KINETICS Software (Beckman Instruments).
Incubation studies using pre-emergent spores.
The effect of polyamine analogues on polyamine synthesis and interconversion was examined on intact pre-emergent spores. Heavy gradient preparations were incubated in W-T medium based on those employed by Weidner & Trager (1973) and Weidner et al. (1999)
in their studies of Nosema excystation and containing 3 mM ATP, 1 mM GTP, 0·5 mM NAD+, 2 mM glucose 6-phosphate, 0·5 mM acetyl-CoA, 1 mM sodium pyruvate, 2 mM glucose in 8 mM NaCl, 138 mM Na3PO4, pH 6·8. We had previously used 150 mM Na3PO4/0·8 % NaCl (Bacchi et al., 2001
), but obtained better uptake and synthesis activity with W-T incubation medium.
Pre-emergent spores from heavy gradient fractions were incubated in the presence of 5 µCi (125 pmol) L-[2,3-3H]ornithine/4 nmol L-methionine for polyamine synthesis and 0·25 µCi (2·1 nmol) [1-14C]spermine for polyamine interconversion in suspensions containing a 250 µl aliquot of cell suspension (100500 µg protein) in W-T medium. Polyamine analogues and radiolabelled substrates were added at the start of the 1 or 2 h incubation period. After incubation, preparations were centrifuged at 12 000 g for 10 min, and pellets were washed in incubation medium and lysed with 250 µl of 10 % (w/v) TCA. Lysates were kept at 04 °C overnight and frozen until analysis. Supernatants from incubations were aspirated from pellets and frozen until analysis. All incubations were done in duplicate.
HPLC analysis.
Lysates and supernatant incubation media from incubations of intact pre-emergent spores with L-[2,3-3H]ornithine or [1-14C]spermine were analysed by HPLC using procedures outlined previously (Bacchi et al., 2001). An IN/US flow-through radiodetector monitored radioactivity and the IN/US
-2 RAM software package (IN/US Systems) was used for data peak integration. Using this system, we obtained the following retention times, based on radiolabelled standards: ornithine, 12 min; putrescine, 24 min; spermidine, 28 min; spermine, 31 min. Acetamidopropionaldehyde had a retention time of 3·5 min, based on UV absorption analysis.
Uptake studies.
Pre-emergent spore preparations were incubated for 15 min in duplicate with varying concentrations of [14 terminal methylenes-14C]spermine, [terminal methylenes-3H(N)]spermidine [1,4-14C]putrescine or L-[2,3-3H]ornithine. The W-T incubation medium was used, with 0·2 ml reaction mixtures centrifuged through 0·2 ml mineral oil at 12 000 g for 0·5 min. Aqueous and oil layers were aspirated, and the tips of microfuge tubes were cut off and analysed by liquid scintillation counting in the presence of Beckman Ready Protein scintillation fluor (Beckman Instruments) as in Goldberg et al. (1998).
Protein determination.
Protein content was estimated by the Bradford method, using BSA as standard.
Chemicals.
Radiolabelled chemicals were purchased as follows: [14 terminal methylenes-14C]spermine (113 mCi mmol1; 4·18 GBq mmol1), Amersham Pharmacia Biotech; L-[2,3-3H]ornithine (40 mCi mmol1; 1·48 GBq mmol1), Moravek Biochemicals; [1-14C]acetyl-CoA (59 mCi mmol1; 2·2 GBq mmol1); [terminal methylenes-3H(N)]spermidine (25 mCi mol1; 0·93 GBq mmol1); [1,4-14C]putrescine (110 mCi mmol1; 4·07 GBq mmol1), NEN Life Science Products.
Polyamine analogues were obtained as follows: SL-11061 (BE 4x4), SL-11093, SL-11144, SL-11158, Dr Benjamin Frydman, SLIL Biomedical (synthesis: Valasinas et al., 2001; Bacchi et al., 2002
); BW-1, Dr Patrick Woster, Wayne State University (synthesis: Zou et al., 2001
). MDL 72521, MDL 72527, MDL 27695, DFMO and difluoromethylarginine were gifts from Marion Merrell Dow. Other chemicals were obtained from Sigma.
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RESULTS |
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Enzymes of polyamine metabolism
Mature spores were broken using the beadbeater technique (Weiss et al., 1994b), clarified by centrifugation and, used as cell-free extracts, assayed for SSAT and PAO, enzymes of polyamine degradation. Results in Table 1
are compared to previously reported data for the synthetic enzymes ODC, SSAT and AdoMetdc (Bacchi et al., 2001
). PAO is four times more active than AdoMetdc and >17-fold more active than ODC. Spermine was the preferred substrate for SSAT, while N1-acetylspermine was the most active substrate for PAO. Other activity/inhibitor data for SSAT and for PAO are given under Effects of polyamine analogues on polyamine metabolism of pre-emergent spore preparations. Arginine decarboxylase activity was less than 0·0036 nmol (mg protein)1 (2 h)1 below the lower limit of detection for the assay. This activity was not inhibitable by difluoromethylarginine (DFMA), a specific inhibitor of the enzyme (Bitonti et al., 1982
). Likewise, DFMA was not an inhibitor of ODC, while DFMO at 1 mM was 55 % inhibitory (Bacchi et al., 2001
).
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We also examined uptake of polyamines in pre-emergent spores to compare rates of uptake. Table 2 lists Km and Vmax values of an experiment in which pre-emergent spores were examined in a rapid uptake study. Incubation mixtures (0·2 ml) containing increasing concentrations of radiolabelled ornithine, putrescine, spermidine and spermine were centrifuged through 0·2 ml mineral oil at 12 000 g. Incubations of 15 min were used in a procedure we have used for uptake studies with African trypanosomes and obtained negligible carry-over of exogenous label (Goldberg et al., 1998
). Table 2
shows that, of the four molecules studied, spermine had the lowest Km value (12·5 nmol) and the highest Vmax [10 000 nmol (15 min)1]. One gauge of the efficacy of transport is the Vmax/Km ratio. For spermine, this ratio was 800, which was >100-fold higher than ratios obtained with data from other substrates. Other preparations yielded similar results.
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Effects of polyamine analogues on polyamine metabolism of pre-emergent spore preparations
In recent work, we had demonstrated that polyamine analogues having a backbone of repeating N-butyl subunits, such as pentamines, oligoamines or bis(aryl)-substituted 3-7-3 analogues (Fig. 1), sterilized Enc. cuniculi-infected monolayer cells and cured two murine model infections (Bacchi et al., 2002
). We began mechanistic studies with some of the compounds that proved to be active in vivo, incubating pre-emergent spore preparations with 8 µM (0·25 µCi) [1-14C]spermine for 1 or 2 h, and examining the resulting 10 % TCA extracts by HPLC and radiodetection for reduction in uptake and interference with spermine interconversion. In an initial series of three experiments, we found that 10 µM SL-11144 and SL-11158 inhibited spermine uptake by 66 % (5977 %) and 63 % (3289 %), respectively. With 0·25 µCi [terminal methylenes-3H(N)]spermidine as substrate, SL-11144 also inhibited uptake 88·5 % (single preparation) and SL-11158 58·8 % (48·668·9 %; two preparations) (data not shown).
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In several experiments, we examined the effects of polyamine analogues on overall spermine uptake, and production of spermidine and acetamidopropionaldehyde. Table 3 gives results of a typical experiment (duplicate incubations) using the analogues SL-11061, SL-11158 and BW-1. In this experiment, we monitored internal (pellet) as well as external (supernatant medium) concentrations of spermine and products. All three analogues caused significant (7080 %) reduction in total spermine uptake, and higher inhibition of spermidine (8295 %) and acetamidopropionaldehyde (7692 %) production. SL-11061 had somewhat lower inhibition of total uptake (69·3 %) as well as lower inhibition of spermidine (82·6 %) and acetamidopropionaldehyde (76·3 %) production. BW-1, although having a median effect on total uptake (73·4 %), inhibited production of both metabolites
90 %. Although little spermidine was found as excretion product, acetamidopropionaldehyde was present in control incubations, and was reduced in SL-11158 (20 %) and BW-1 (27 %) supernatants. If incubations continued for 2 h, supernatant media of analogue-treated cells had 3·2- to 11·5-fold more spermine remaining in the medium than controls incubated without analogues (not shown), indicating that less uptake was occurring in these cells.
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To determine the effect of SL-11158 on enzyme kinetics of Enc. cuniculi SSAT, we measured enzyme activity in the presence of none, 0·54 and 1·08 mM SL-11158 with increasing concentrations of spermine (2·7, 5·4 and 10·8 mM). Incubations contained 100 µM Bicine buffer, pH 8·0, 17 µM (60 µCi mmol1) [1-14C]acetyl-CoA, inhibitor and spermine and 114 µg Enc. cuniculi spore protein preparation. After 30 min, reactions (in triplicate) were spotted on filter paper discs, washed and counted in Ominfluor. Blank reactions without spermine and protein were also run and subtracted. Results were analysed using a HanesWoolf primary plot (V/[S] versus substrate concentration). A Km value of 17·3 mM was obtained for spermine and a Ki value of 0·24 mM was obtained for SL-11158 from a secondary HanesWoolf plot of Km versus inhibitor concentration (Fig. 2). This is a mixed type of inhibition in which the inhibitor has an affinity for the enzyme which is much higher than that of the substrate.
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The pH optima of the enzymes also differed: pH 10·0 for mammalian PAO and pH 7·08·0 for the protozoan enzyme (Table 4). FAD superadded to control reaction mixtures did not stimulate the rate of the Enc. cuniculi enzyme. Two enzyme-activated irreversible inhibitors of mammalian PAO were developed by Merrell Dow Research Laboratories, MDL 72521 and MDL 72527 (Bey et al., 1985
). These exhibited time- and dose-dependent kinetics and had Ki values of 0·10·3 µM for the mammalian enzyme (Seiler, 1987
). These analogues, used at up to 100 µM, stimulated the Enc. cuniculi enzyme by up to 70 % (Table 5
).
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None of the SLIL amine analogues studied (SL-11061, SL-11093, SL-11144, SL-11158) served as substrate for the Enc. cuniculi PAO at 100 µM, and only SL-11144 and SL-11158 were significant inhibitors of the reaction at 100 µM (75 and 57 %, respectively). MDL 27695 (1 mM) was a substrate, having 34 % of the activity with the natural substrate, while BW-1 (1 mM), a bis(phenylbenzyl) 3-7-3 analogue of MDL 27695, had 55 % of control activity, and stimulated the control reaction (with N1-acetylspermine as substrate) by 40 %. Pargyline is an inhibitor of monoamine oxidases, but inhibited the full reaction by 80 % and did not affect the mammalian enzyme (Table 5). These analogues did not affect the horseradish peroxidase reaction with H2O2 as substrate.
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DISCUSSION |
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Similarly, the ability of Enc. cuniculi to take up spermine and convert it to spermidine and putrescine, coupled with the high efficacy of spermine uptake as opposed to other polyamines or precursors, makes it likely that this microsporidian utilizes polyamine salvaged from the host cell as a major mechanism of acquiring polyamines. The kinetics of transport indicate it is used by Enc. cuniculi to scavenge any available polyamines from the host cytoplasm. Other protozoan parasites such as Trypanosoma cruzi and Trichomonas vaginalis are also believed to obtain most of their polyamines via exogenous sources (Le Quesne & Fairlamb, 1996; Ariyanayagam & Fairlamb, 1997
; Yarlett et al., 1994
). Since spermine uptake was not blocked by monensin, gramicidin S, ouabain or amiloride, but was inhibited by valinomycin, it appears that Na+/K+ ATPase was not involved in uptake, but a universal cation transporter may be responsible. Relatively low inhibition (31 %) of transport obtained with KCN reinforces the finding that the microsporidia have neither mitochondria nor a functional cytochrome system (Undeen, 1990
).
The high activity of exogenous spermine uptake in pre-emergent spores, and the increasing focus on polyamine analogues as anti-tumour agents (Casero & Woster, 2001; Frydman & Valasinas, 1999
), made it imperative that we examine analogues as anti-microsporidial agents (Bacchi et al., 2002
). In mammalian cells, these are taken up through polyamine transporters and down-regulate polyamine synthesis while inducing SSAT and promoting excretion of acetylated polyamines. As polyamine levels fall in treated cells, the concentrations of analogues increase. Since they do not function in cell metabolism as do polyamines, division stops and apoptosis begins (Casero & Woster, 2001
; Frydman & Valasinas, 1999
; Ha et al., 1998
).
In the present study, we worked with pre-emergent spore fractions, which, although relatively homogeneous, were not actively dividing cells. Thus, effects of analogues seen on dividing mammalian cells such as SSAT induction, reduction in ODC and AdoMetdc activities, could not be readily observed. However, these analogues had several definitive effects on polyamine metabolism of Enc. cuniculi, including reduction of spermine uptake and interconversion (SL-11144, SL-11158) and mixed inhibition of SSAT (SL-11158). BW-1 was a substrate for PAO and in vivo is thus a likely competitive inhibitor of the enzyme with N1-acetylspermine as substrate. The related analogue MDL 27695 [a bis(benzyl) 3-7-3 analogue; Fig. 1] is debenzylated by mammalian PAO (Bitonti et al., 1990
). MDL 27695 at 1 mM had modest (33 %) substrate activity in the Enc. cuniculi PAO assay. BW-1, a bis(phenylbenzyl) 3-7-3 analogue, had higher substrate activity (54·9 %) in this system, and, used in the presence of the natural substrate, stimulated enzyme activity, by 2·6-fold (Table 5
).
These studies indicate that Enc. cuniculi has significant polyamine scavenging potential with spermine the preferred substrate and it is likely that interconversion of polyamines readily occurs in this intracellular parasite. SL-11144, SL-11158 and BW-1 interfere with polyamine uptake and interconversion in the intact pre-emergent spores, while SL-11158 gives a mixed type of inhibition of parasite SSAT. BW-1 is a substrate for parasite PAO and NMR studies in progress indicate two products are formed from [13C]BW-1, a carboxylic acid and an aldehyde, which is consistent with an oxidation pathway (P. Woster, personal communication). All these effects occur at similar analogue concentrations, indicating that the mechanism of action of these agents in vivo targets this pathway.
Since existing chemotherapeutic agents for the microsporidia are not universally curative in human infections (Wittner & Weiss, 1999), more effective agents are needed. Continuing studies are aimed at developing more active agents by examining structureactivity relationships and clarifying mechanism(s) of action.
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ACKNOWLEDGEMENTS |
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Received 5 November 2003;
revised 7 January 2004;
accepted 15 January 2004.