Leishmania species: Evidence for transglutaminase activity and its role in parasite proliferation

Experimental Parasitology 114 (2006) 94–102 www.elsevier.com/locate/yexpr

Leishmania species: Evidence for transglutaminase activity and its role in parasite proliferation Reynolds K.B. Brobey a, Lynn Soong a,b,¤ a

Department of Microbiology and Immunology, Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555-1070, USA b Department of Pathology, Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555-1070, USA Received 5 January 2006; received in revised form 18 February 2006; accepted 20 February 2006 Available online 18 April 2006

Abstract Albeit transglutaminase (TGase) activity has been reported to play crucial physiological roles in several organisms including parasites; however, there was no previous report(s) whether Leishmania parasites exhibit this activity. We demonstrate herein that TGase is functionally active in Leishmania parasites by using labeled polyamine that becomes conjugated into protein substrates. The parasite enzyme was about 2- to 4-fold more abundant in Old World species than in New World ones. In L. amazonensis, comparable TGase activity was found in both promastigotes and amastigotes. TGase activity in either parasite stage was optimal at the basic pH, but the enzyme in amastigote lysates was more stable at higher temperatures (37–55 °C) than that in promastigote lysates. Leishmania TGase diVers from mouse macrophage (M) TGase in two ways: (1) the parasite enzyme is Ca2+-independent, whereas the mammalian TGase depends on the cation for activity, and (2) major protein substrates for L. amazonensis TGase were found within the 50–75 kDa region, while those for the M TGase were located within 37–50 kDa. The potential contribution of TGase-catalyzed reactions in promastigote proliferation was supported by Wndings that standard inhibitors of TGase [e.g., monodansylcadaverine (MDC), cystamine (CS), and iodoacetamide (IodoA)], but not didansylcadaverine (DDC), a close analogue of MDC, had a profound dose-dependent inhibition on parasite growth. Myo-inositol-1-phosphate synthase and leishmanolysin (gp63) were identiWed as possible endogenous substrates for L. amazonensis TGase, implying a role for TGase in parasite growth, development, and survival. © 2006 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Leishmania; Trypanosomatid; Parasite; Promastigote; Amastigote; Western blot; Inhibition; Transglutaminase (EC 2.3.2.13); Glycoconjugates; TGase, transglutaminase; MDC, monodansylcadaverine; DDC, didansylcadaverine; CS, cystamine; IodoA, iodoacetamide; FC, Xuorescein cadaverine; PTM, posttranslational modiWcation; MES, 2-[N-morpholino]ethanesulfonic acid; M, macrophage; BCA, bicinchonic acid

1. Introduction Leishmaniasis is a major parasitic disease for which no eVective vaccine is available. Chemotherapy relies predominantly on pentavalent antimonials, but resistance to them is increasing yearly (Handman, 2001). Moreover, toxicity associated with their use has become a major concern

*

Corresponding author. Fax: +1 409 747 6869. E-mail address: [email protected] (L. Soong).

0014-4894/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2006.02.021

(Handman, 2001), and the search for new therapeutic targets based on the parasite biology remains a priority. Posttranslational covalent modiWcations (PTMs) have long been associated with Leishmania survival and infectivity. In recent years, several PTM-modiWed proteins and molecules involved in their synthesis have been identiWed (Davis et al., 2004; Descoteaux and Turco, 1999; Ilg, 2001; Poonam et al., 2000). While phosphorylations (Poonam et al., 2000) and glycosylations (Descoteaux and Turco, 1999; Ilg, 2001) have been described in Leishmania, the potential contribution of PTM to Leishmania biology involving the formation of isopeptide bonds catalyzed by

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transglutaminases (TGases: EC 2.3.2.13) has not been investigated. TGases are family of enzymes that insert an irreversible isopeptide bond within and/or between proteins by using speciWc glutamine residues on one protein and the primary amine group on the other molecule (Greenberg et al., 1991; Lorand and Conrad, 1984). The resultant PTM molecules are resistant to proteinases and denaturants (Greenberg et al., 1991). The enzyme activity from several sources is Ca2+-dependent; however a number of Ca2+-independent isoforms have also been found (Adini et al., 2001; Del Duca and SeraWni-Fracassini, 2005; Pasternack et al., 1998). The reason cells need protein cross-links catalyzed by TGases continues to puzzle investigators, although the signiWcant contribution of the enzyme in blood clotting and wound healing is well documented (Akimov and Belkin, 2001; Verderio et al., 2005). Further, TGase-catalyzed cross-links have been observed in diverse cellular processes, including growth and diVerentiation, cellular adhesion and receptormediated endocytosis, and in the general upholding of tissue integrity (Akimov et al., 2000; Nicholas et al., 2003). Leishmania alternates between two developmental forms: promastigotes reside mainly in the gut of sand Xies (where pH could be slightly alkaline (Gontijo et al., 1998) and temperature is slightly above 20 °C), but interact brieXy with the mammalian host at the early stages of infection (Matlashewski, 2001). On the other hand, amastigotes infect cells of the macrophage lineage and replicate in the phagolysosomes [where the resident pH is acidic (Mukkada et al., 1985) and the temperature is close to 37 °C]. These two disparate ecological habitats present enormous challenges to the invading parasite, as it must overcome a variety of toxic substances and avoid detrimental exposure to host hydrolytic enzymes. Species-speciWc, parasite-derived glycoconjugates are known to protect Leishmania against hydrolytic enzymes and facilitate parasite attachment to the insect midgut (Matlashewski, 2001; Russel, 1994). In HIV, the envelope glycoconjugates gp120 and gp41 are endogenous substrates for viral TGase (Mariniello et al., 1993a,b). It is possible that Leishmania may utilize TGase cross-links to ensure its survival within the host. There is currently no report on TGase-like sequences in the Leishmania protein database, in spite of the completion of L. major Friedlin genome sequencing, implying that Leishmania TGase, if its identity could be veriWed, is very diverge structurally from other known TGases. In this study, we demonstrate that Leishmania parasites exhibit TGase activity that is mechanistically diVerent from TGase of the mouse macrophage (M). Further enzymatic analyses of the parasite lysates showed that Leishmania TGase activity resembles TGase of other microorganisms in terms of its sensitivity to inhibitors, pH, and temperature, and ability to incorporate labeled primary amine into proteins. We also demonstrate the relevance of this activity to parasite proliferation. To the best of our knowledge, this is the Wrst report of a functional TGase in Leishmania parasites.

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2. Materials and methods 2.1. Chemicals and reagents Cystamine (CS), monodansylcadaverine (MDC), didansylcadaverine (DDC), iodoacetamide (IodoA), recombinant human TGase II, and protease inhibitors were purchased from Sigma (St. Louis, MO). Dimethylcasein and streptavidin-conjugated alkaline phosphatase were products of EMD Biosciences (La Jolla, CA). Fluorescein cadaverine was from Molecular Probes (Eugene, OR), while mouse anti-Xuorescein horseradish peroxidase was purchased from CHEMICON International (Temecula, CA). 5-(Biotinamido) pentylamine was obtained from Pierce ScientiWc (Rockford, IL). 2.2. Parasites L. amazonensis (LV78) axenic amastigotes were kindly provided by Dr. K.-P. Chang of Rosalind Franklin University of Medicine and Science. Parasites were routinely cultured at 32 °C under 5% CO2 in Grace’s Insect growth medium (Gibco-BRL, Rockville, MD), pH 5.3, supplemented with 20% FBS (Sigma) and 2 mM L-glutamine. The promastigote stage of LV78 was generated from axenic amastigotes. In addition, the following promastigotes were used: L. amazonensis (MHOM/BR/77/LTB0016), L. major (MRHO/SU/59/P/LV39) obtained from Dr. Richard Titus of Colorado State University, Fort Collins, L. major MHOM/IL/80/Friedlin (FN) obtained from Dr. Phillip Scott of University of Pennsylvania School of Veterinary Medicine, Philadelphia, and L. chagasi (MHOM/CO/84/ C1044) provided by Dr. Mary Wilson of University of Iowa. Promastigotes were cultured in Schneider’s Drosophila growth medium (Life Technologies, Rockville, MD), pH 7.0, supplemented with 20% FBS, 2 mM L-glutamine, and 50 g/ml gentamicin. To maintain the infectivity of the parasites, BALB/c mice were infected subcutaneously in the hind foot with 2 £ 106 stationary-phase promastigotes, and lesion-derived parasites were recovered at 6–10 weeks postinfection. Mice were purchased from Harlan Sprague– Dawley (Indianapolis, ID), maintained under speciWc pathogen-free conditions, and used at 6–7 week of age under protocols approved by the Animal Care and Use Committee of the University of Texas Medical Branch, Galveston, Texas. 2.3. Parasite lysates Parasites (1–3 £ 109) were washed three times in PBS and suspended in freshly prepared lysis buVer containing 0.1 M Tris–Cl, pH 8.0, 0.15 M NaCl, 10 mM DTT, 2% Triton X-100, and a protease inhibitor cocktail. Samples were freeze/thawed three times, sonicated for a total of 3 min (30 s per round) on ice using a Braun-Sonic model 1000L equipped with 40TL probe. Lysed cells were then centrifuged at 20,000g for 15 min. Clear lysates were dialyzed

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against the same buVer overnight using a 4-kDa cut-oV membrane to remove contaminating endogenous polyamines. Aliquots from each preparation were then diluted for protein concentration determination using a BCA protein assay. Lysates were either used the same day or immediately frozen down in aliquots at ¡70 °C until analysis. 2.4. Mouse macrophage Bone marrow-derived macrophage (M) cultures were generated as previously described (Soong et al., 1996). BrieXy, mouse femur-derived bone marrow cells were cultured in Petri dishes (15 £ 100 mm) at a concentration of 2 £ 106 cells per dish in Iscove’s modiWed Dulbeco’s media (IMDM; Gibco) supplemented with 2 mM L-glutamine, 10% FBS, 50 g/ml gentamycin, 1 mM sodium pyruvate, 50 M 2-mercaptoethanol (Sigma), 100 U/ml penicillin, 10 g/ml streptomycin, and 10% L-929 cell supernatant. Upon 5 days in culture, non-adherent cells were discarded, and the adherent cells were maintained in complete IMDM containing 5% L-929 cell supernatant. On day 7, cells were detached with cold 2 mM EDTA/PBS, washed, and plated into 24-well tissue culture plates at a concentration of 3 £ 105 cells per well. For protein preparation, adherent M (»2.2 £ 108) were lysed by sonication for 3 min using Braun-Sonic model 1000L equipped with 40TL probe as described above and centrifuged at 25,000g for 20 min. The clear lysates were subsequently dialyzed overnight as described in Section 2.3. 2.5. Microwell plate assay for TGase activity TGase activity was routinely determined in a microtiter plate using a modiWed protocol originally described elsewhere (Slaughter et al., 1992). The microwell plates were coated with dimethylcasein (30 mg/ml) overnight at 4 °C; uncoated sites were blocked with 5% non-fat dried milk in Tris-buVered saline containing 0.05% Tween 20 (TBST). The reaction mixture made in 200 l contained 100 mM Tris–HCl, pH 8.5, 10 mM DTT, 1 mM 5-(biotinamido) pentylamine, 1.8 mg/ml protein lysates, and the presence or absence of 5 mM CaCl2. The reaction was performed at 37 °C for 30 min, and TGase-catalyzed conjugation of amine donor 5-(biotinamido) pentylamine into dimethylcasein was determined by streptavidin-alkaline phosphatase and p-nitrophenol phosphate reporter system at 405 nm. Recombinant human TGase II (5 ng/ml) was used as a positive control to normalize the activity in each assay.

stability/activity was determined by pre-incubating parasite lysates for 20 min at temperatures ranging over 25–65 °C, and assaying for TGase activity at 25 or 37 °C. 2.7. IdentiWcation of endogenous substrates for TGase Parasite or macrophage lysates (0.8–1.5 mg/ml) were incubated with 4 mM Xuorescein cadaverine (FC) for 4 h at 37 °C with constant agitation in 24-well culture plates. Thereafter, reaction products were treated with equal volumes of Laemmli sample buVer and loaded onto 10% SDS–PAGE (BioRad Criterion gels). Separated proteins were electroblotted onto PVDF membrane, and conjugation of FC into various proteins catalyzed by TGase was revealed using horseradish peroxidase-conjugated Xuorescein antibody and Visualizer™ Western blot detection system (UPSTATE, Lake Placid, NY). Replicate SDS–PAGE gels were stained with Coomassie SimplyBlue (Invitrogen, Carlsbad, CA), and corresponding protein bands were excised and subjected to mass spectrometry (LC–MS/MS) identiWcation at the University of Texas Medical Branch Proteomics Core Facility. 2.8. Growth inhibition assay and reversibility studies Leishmania amazonensis promastigotes were cultured in complete Schneider’s Drosophila medium for 3 days, as described in Section 2.2. The culture was then diluted 100fold (»105 cells) with fresh medium into 24-well culture plates pre-dosed with TGase inhibitors at the following concentration ranges: 0–5 mM cystamine (CS), 0–0.5 mM monodansylcadaverine (MDC), and 0–0.1 mM iodoacetamide (IodoA). Didansylcadaverine (DDC), a substituted analogue of MDC, was used at 0–0.4 mM as a control chemical. Parasites were cultured continuously in the presence or absence of the inhibitors for 72 h. Thereafter residual viable parasites were harvested and counted microscopically. Cell viability was scored by mobility, as well as by the ability of live parasites to exclude trypan blue dye. Triplicate wells were done for each inhibitor concentration and results were averaged for two independent experiments. The reversible inhibitory action of MDC on promastigotes was investigated by culturing parasites in replicate in the presence of 0.15 mM of the inhibitor. Cultures were monitored for parasite growth and morphological changes daily. At day 3, a group of cultures were washed oV the inhibitor and replaced with fresh medium without the drug. Aliquots of cultures were withdrawn daily as before for counting parasite numbers and morphological changes.

2.6. EVect of pH and temperature on TGase activity 3. Results The eVect of pH on TGase activity in the promastigote or amastigote lysates was investigated using diVerent buVers ranging over pH 5.5–10.5. The buVers employed were MES (0.1 M, pH 5.5), phosphate (0.1 M, pH 6 and 7), Tris–HCl (0.1 M, pH 7.5, 8.5, and 9.5), and glycine–NaOH (0.1 M, pH 10.5). The eVect of temperature on the enzyme

3.1. Detection of Ca2+-independent TGase activity in Leishmania parasites TGase activity was examined in promastigote lysates of L. major strains FN and LV39, L. amazonensis strains

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Table 1 Colorimetric determination of TGase activity in various Leishmania species and strains Leishmaniaa

Geographical distribution

L. major LV39 FN

Old World

L. amazonensis LTB0016 LV78

New World

L. chagasi C1044

New World

TGase activity (U/mg protein)b 54.52 § 1.36 80.18 § 2.10 24.66 § 2.82 21.58 § 0.58 21.54 § 1.86

a

Proteins were extracted from parasites, dialyzed to remove endogenous inhibitors of TGase, and quantitated by the BCA protein assay. b Data represent average value for triplicate of reaction and deviations are shown (§SD). Unit (U) is deWned as amount of pentylamine conjugated into dimethylcasein per minute, monitored by the streptavidin-alkaline phosphatase and p-nitrophenol reporter system at 405 nm.

Fig. 1. TGase activity in Leishmania lysates is subject to inhibition. Dialyzed parasite lysates were quantitated by the BCA protein assay, and »1.8 mg/ml proteins from each parasite stage was used for TGase activity measurement using the standard microtiter plate assay. TGase activity in promastigotes (Wlled bars) or amastigotes (open bars) was determined in the absence or presence of TGase inhibitors iodoacetamide (IodoA), cystamine (CS), and monodansylcadaverine (MDC). Heat-inactivated lysates were included as a control. TGase activity without the inhibitor was taken as 100%, and was approximately identical for both amastigotes and promastigotes when reaction was performed at 37 or 25 °C, respectively, representing optimal temperature conditions for the parasite stages. At least two independent experiments were performed on each extract, and shown are the averaged values for triplicate reactions of a representative experiment (means § SD).

LTB0016 and LV78, and L. chagasi, by the covalent incorporation of biotinylated pentylamine into dimethylcasein in the presence of CaCl2 used in a standard TGase assay. As summarized in Table 1, TGase-speciWc activity was about 2- to 4fold higher in L. major (Old World) than those in L. amazonensis and L. chagasi (New World). TGase activity was subsequently examined in lysates of LV78 amastigotes and promastigotes in the presence or absence of TGase inhibitors.

Fig. 2. Leishmania TGase does not require Ca2+. TGase activity was determined in L. amazonensis promastigotes, alongside mouse M or human TGase II, in the absence or presence of 5 mM CaCl2. Leishmania TGase and mouse macrophage TGase were from total lysates; recombinant human TGase II was obtained commercially. Data represent means § SD of two independent experiments.

As shown in Fig. 1, comparable TGase activity was demonstrable in amastigote lysates, and, like the enzyme in promastigotes, was sensitive to inhibition in a dose-dependent manner. MDC was most eVective, inhibiting about 90% of the activity in promastigotes and 70% in amastigotes at 0.5 mM. CS at 10 mM inhibited more than 75% of TGase activity from both lysates, while IodoA at 10 mM inhibited »70% of promastigote and 50% of amastigote TGase, respectively (Fig. 1). By contrast, no covalent incorporation of pentylamine by TGase was observed for amastigote or promastigote lysates that were previously heat-inactivated at 75 °C for 20 min (Fig. 1). To examine whether Ca2+ is required for Leishmania TGase activity, dialyzed parasite lysates were assayed for TGase activity, alongside mouse M lysates and recombinant human TGase II, in the presence or absence of 5 mM CaCl2. A representative proWle is shown in Fig. 2. While Leishmania TGase remained catalytically active in the absence of Ca2+, with mouse M TGase, like its human counterpart (TGase II), lost activity entirely when Ca2+ was excluded from the reaction mixture. Moreover, inclusion of EGTA to chelate endogenous Ca2+ in the reaction mixture did not aVect enzymatic activity of the Leishmania TGase. 3.2. Dependence of Leishmania TGase activity on temperature and pH In nature, promastigotes reside mostly in the insect gut where pH could be slightly alkaline (Gontijo et al., 1998) and temperature is moderate, i.e., above 20 °C, whereas, amastigotes live exclusively in mammalian phagolysosome where the resident pH is acidic (Mukkada et al., 1985) and the temperature is close to 37 °C. To investigate if these physical parameters inXuence the catalysis of the parasite

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Fig. 3. Leishmania TGase temperature tolerance is stage-dependent. Lysates of promastigotes (A) and amastigotes (B) were pre-incubated for 20 min at the temperatures 25, 37, 45, 55, and 65 °C, and quickly chilled on ice. Thereafter, residual TGase activity was determined at either 25 °C (open bars) or 37 °C (Wlled bars) by a microtiter plate assay.

enzyme at various life stages, the eVect of temperature and pH on Leishmania TGase was investigated. As shown in Fig. 3, Leishmania TGase sensitivity to temperature was stage-dependent. When promastigote or amastigote lysates were pre-incubated at temperatures ranging from 25 to 65 °C for 20 min and assayed for TGase activity at 25 or 37 °C, the amastigote enzyme (Fig. 3B) tolerated higher temperatures (37–55 °C) than the enzyme in promastigote lysates (Fig. 3A). The amastigote TGase was maximally active at 37–45 °C, while optimal activity for the promastigote enzyme occurred at 25 °C. Nevertheless, both enzymes completely lost activity beyond 65 °C. The leishmanial TGase response to pH, however, was identical in both parasite forms (data not shown). When TGase activity was determined at pH 5.5, 6, 7, 7.5, 8.5, 9.5, and 10.5 in promastigote or amastigote lysates, the TGase activity in either parasite form was optimal at basic pH ranges (8.5–9.5). 3.3. IdentiWcation of potential TGase protein substrates on Western blots To further demonstrate the presence of TGase activity in Leishmania that is diVerent from the host, we examined possible endogenous substrate(s) for the enzyme in L. amazonensis promastigotes and amastigotes, and mouse M using labeled amine donor FC. As shown in Fig. 4, protein labels were found only in CS-minus lanes, while in the

Fig. 4. TGase-catalyzed incorporation of Xuorescein cadaverine into protein substrates. Leishmania or M lysates were incubated with 4 mM Xuorescein cadaverine (FC) in the presence or absence of 15 mM cystamine (CS). The FC-treated proteins were then separated by SDS–PAGE and transferred onto PVDF membranes. Bound FC was revealed by horseradish peroxidase-conjugated anti-Xuorescein antibody. Major endogenous substrates for Leishmania TGase were found within 50–75 kDa, and were more abundant in promastigotes (Pro) than in amastigotes (Am). Major endogenous substrates of mouse M were within 37–50 kDa. Filled arrows indicate the positions of four resolved signals in amastigotes that were excised for identiWcation by LC–MS/MS: (a) leishmanolysin (68 kDa) and (b) myo-inositol-1-phosphate synthase (58 kDa); (c) and (d) were not accessible in the Leishmania database. The open arrow shows the predominant »45 kDa substrate(s) from mouse M.

presence of the inhibitor, no FC conjugation was observed. A broad 45-kDa band was prominently labeled for M lysates (open arrow), while a 65-kDa signal was consistently detected in parasite lysates, with much higher intensity in promastigotes than in amastigotes (Fig. 4). An additional three labeled bands in promastigotes, and two in amastigotes in the range 20–37 kDa were observed. A signal of »55 kDa was further detected in amastigote blots. The amastigote blot was matched with an identical protein gel stained with Coomassie dye, and the four separate bands (denoted a–d) were excised for protein identiWcation via LC–MS/MS. The band (a) was identiWed as L. chagasi homologue of leishmanolysin, a 68.7-kDa protein predominantly expressed on the surface of promastigotes (also called promastigote surface endopeptidase), whereas the band (b) was mapped to L. amazonensis myo-inositol-1phosphate synthase, a 58.5-kDa protein whose function includes the synthesis of inositol. Bands (c) and (d) could not be conWdently identiWed from the Leishmania database. 3.4. TGase inhibitors reduce promastigote proliferation Having demonstrated the inhibition of TGase activity in Leishmania lysates, we then determined whether the

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inhibitors of TGase would aVect promastigote proliferation. Parasites were cultured continuously for 72 h in the presence or absence of TGase inhibitors, and viable parasites counted microscopically. As shown in Fig. 5, promastigote growth was profoundly inhibited in a dose-dependent manner by all three tested inhibitors. The eVective concentration required to achieve 50% inhibition (EC50) was about 0.003 mM for IodoA (Fig. 5C), 0.15 mM for MDC (Fig. 5B), and 3.0 mM for CS (Fig. 5A). In contrast, DDC, a substituted analog of

99

MDC, had no inhibitory action on parasite growth (Fig. 5B), even when it was used at 0.5 mM (data not shown). 3.5. MDC reversibly suppresses promastigote growth and morphological development MDC is a substrate analogue, and a competitive inhibitor of TGases. To investigate whether MDC action on promastigotes observed in this study may be attributable to non-speciWc toxicity, we conducted reversibility studies on MDC-treated parasites. As shown in Fig. 6A, in the presence of 0.15 mM MDC, parasites still showed signs of growth within the Wrst 24 h (arrow a), and were morphologically normal (Fig. 6B, a). Growth began to decrease at days 2 and 3 (Fig. 6A, arrows b and c), at which time a majority of parasites became disk-shaped, but motile promastigotes (Fig. 6B, b and c). Parasites that were stripped of the inhibitor at day 3 resumed growth at day 5 (Fig. 6A) and thereafter (arrows d and e) attained the long, slender shape that is typical of mature promastigotes (Fig. 6B, d or e). Parasites that remained with the inhibitor from days 3 to 14, however, were still disk-shaped morphologically, until eventually their numbers dropped beyond the limit of detection possible by microscopy (Fig. 6A). The results appear to suggest that MDC, by inhibiting the target TGase in the parasite, impairs the parasite ability to replicate, and to mature to the elongated shape characteristic of infective promastigotes. 4. Discussion

Fig. 5. TGase inhibitors reduce promastigote proliferation in vitro. Promastigotes (1 £ 105 cells/well) were grown in 24-well culture plates for 72 h in the absence or presence of TGase inhibitors at the indicated concentrations. Thereafter, residual parasites were harvested and counted under a light microscope. Each point on the graph represents a triplicate of culture and was averaged for two independent experiments. The culture without inhibitor showed an over 5-fold growth relative to starting parasites. (A) Cystamine; (B) MDC, monodansylcadaverine; DDC, didansylcadaverine; (C) iodoacetamide. Note: iodoacetamide also inhibits cysteine proteinase.

TGases are a functionally ubiquitous group of enzymes that insert irreversible isopeptide bonds within and/or between proteins, leading to their posttranslational modiWcations (PTMs) (Greenberg et al., 1991; Lorand and Conrad, 1984). TGase-catalyzed PTMs have been implicated in several cellular processes, including wound healing (Verderio et al., 2005), and the enzyme has been utilized in industries to treat fabric wools against detergent damage (Cortez et al., 2005). Although TGases continue to attract attention in tumor research and in some infectious diseases [for review, see GriYn et al. (2002)], only a few parasite enzymes have been identiWed to date (Davids et al., 2004; Mehta et al., 1996; Rao et al., 1999). Using a quantitative colorimetric assay and a qualitative Western blot, we have shown for the Wrst time that Leishmania has TGase activity that is more abundant in the Old World species than in those of the New World. In L. amazonensis, TGase activity was detected in both promastigotes and amastigotes. TGase activity in amastigotes tolerated higher temperatures, ranging from 37 to 55 °C, while the promastigote enzyme had limited activity at these temperatures. The observed diVerences in sensitivity to temperature may reXect the diVerent temperatures in the environment where both parasite stages are found (Matlashewski, 2001). This feature of high temperature tolerance, however, is not unique to the amastigote enzyme. TGase from a Wlarial worm Brugia

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Fig. 6. MDC reversibly suppresses promastigote proliferation and morphological development. Promastigotes were cultured in replicates in the presence of 0.15 mM MDC. At day 3, a group of replicate cultures was washed to remove the inhibitor and replaced with fresh medium without the drug. Aliquots of cultures were harvested at the indicated time points for counting parasite numbers (A) and examination of morphological changes (a–e), (B).

malayi was catalytically active at 50–70 °C (Singh and Mehta, 1994). As found with TGases of Wlarial parasites and Bacillus subtilis (Singh and Mehta, 1994; Suzuki et al., 2000), the leishmanial TGases from both promastigotes and amastigotes were optimally active at basic pH (8.5–9.5). Unlike most known TGases, including TGase from mouse M lysate, Leishmania TGase activity does not require Ca2+. Reported Ca2+-independent TGases include those identiWed from Plasmodium falciparum (Adini et al., 2001), rat intestine (Tsai et al., 1998), plants (Del Duca and SeraWni-Fracassini, 2005), and some bacteria (PXeiderer et al., 2005). The relatively low Ca2+ levels in environments inhabitable by Leishmania (Philosoph and Zilberstein, 1989; Pollack et al., 1986) might explain the Ca2+-independency of Leishmania TGase. The eVect of inhibitors on Leishmania TGase activity is consistent with the inhibition of TGase from other parasites (Adini et al., 2001; Rao et al., 1991; Singh and Mehta,

1994). Our Wndings that the chemicals MDC, CS, and IodoA inhibited leishmanial TGase and were equally eVective against parasite growth, underscores the relevance of TGase activity in Leishmania replication. An enzyme– inhibitor interaction was speciWc, since closely related chemical DDC, a substituted analog of MDC, had little or no inhibitory eVect on parasite growth. In addition, the suppressive eVect of the competitive inhibitor MDC on promastigotes was reversible, implying speciWc interactions between the inhibitor and its target protein(s). Although MDC-treated parasites showed gross growth impairment, the eVect of the chemical on parasite development needs further investigation. We attempted to identify protein substrates for Leishmania TGase using immunoblotting and mass spectrometry and found »58 and 68-kDa labeled proteins with very high homology to L. amazonensis myo-inositol-1-phosphate synthase (I-1-P synthase) and L. chagasi leishmanolysin

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(gp63), respectively. An I-1-P synthase is involved in the primary synthesis of inositol, which is a component of glycoconjugates. Since viral glycoconjugates gp20 and gp41 are documented substrates for viral TGase (Mariniello et al., 1993a,b), studies of Leishmania TGase-I-1-P synthase interaction will further strengthen our understanding of the roles of glycoconjugates in Leishmania biology. Interestingly, gp63 is a well-known Leishmania glycoconjugate that plays crucial roles in protecting both promastigotes and amastigotes against hydrolytic enzymes in their respective habitats (Mottram et al., 1992; Russel, 1994). Besides, gp63 has been implicated in promoting Leishmania uptake by macrophages (Bart et al., 1997). Since parasite interaction with macrophages and subsequent uptake are multifunctional ventures, gp63-TGase association, like I-1-P synthase interaction with Leishmania TGase, is an important area for further study. Indeed, TGase involvement in retonic acid-induced Trypanosoma cruzi interaction with and uptake by macrophages has been suggested (Wirth and Kierszenbaum, 1986). Our accumulated data overwhelmingly support the existence of TGase activity in Leishmania parasites. Moreover the activity, undoubtedly, is important for the parasite, an observation that warrants continued investigation in terms of isolating the protein(s) responsible for the enzymatic activity. To this end, we have initiated schemes to purify TGase from promastigote lysates. Presently, we denote this activity as TGase-like and cogitate on its possible roles in the two life stages of Leishmania. Although there is limited information regarding interactions of Leishmania parasites with the insect vector, it has been demonstrated that the attachment of promastigotes to the insect gut is facilitated by parasite-derived glycoconjugates (Mottram et al., 1992). Whereas glycoconjugates’ cross-interaction with the insect proteins would promote adherence, further investigation of the involvement of parasite TGase is expected to advance our understanding of these associations, since the extracellular activity of TGase has been shown to facilitate cell attachment (Aeschlimann and Thomazy, 2000). Similarly, the extracellular activity of TGase would promote amastigote–macrophage interactions and subsequent internalization, as was suggested in the case of T. cruzi-macrophages (Wirth and Kierszenbaum, 1986). In conclusion, the present study utilizes colorimetric activity determination and immunoblotting to demonstrate the presence of functional TGase in Leishmania parasites. Growth inhibition assay further implicates the Leishmania TGase in parasite replication. Since fundamental diVerences appear to exist between the host and the parasite enzymes, further characterization of these diVerences will reveal new potential targets in Leishmania for therapeutic intervention. Accordingly, the present study further increases our knowledge in Leishmania biology. Acknowledgments We wish to thank Nanchaya Wanasen for technical support with mouse M, and Mardelle Susman for reading the

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