Eur. J. Biochem. 225, 333-339 (1994) 0 FEBS 1994

Trans-sialidase and sialidase activities discriminate between morphologically indistinguishable trypanosomatids

Enrique MEDINA-ACOSTA', Antonia M. R. FRANCO', Ana Maria JANSEN'.3, Marcos SAMPOL2, Neuza NEVES'.', Lain PONTES-DE-CARVALHO 1,4,Gabriel GRIMALDI, Jr2 and Victor NUSSENZWEIG' ' New York University Medical Center, Michael Heidelberger Division of Immunology, New York, USA * Instituto Oswaldo Cruz, Rio de Janeiro, Brazil Instituto de CiSncias da Salide, UFBA, Brazil Centro de Pesquisas Gonqalo Moniz, Fundaqao Oswaldo Cruz, Brazil

(Received June 2/August 12, 1994) - EJB 94 0795/4

The expression of trans-sialidase and sialidase activities in the kinetoplastid protozoa was ex- plored as a potential marker to discriminate between the morphologically indistinguishable flagel- lates isolated from human, insects and vertebrate reservoir hosts. By virtue of the differences ob- served in the ratios of these enzyme activities, a collection of 52 species and strains comprising the major taxa of these parasites could be separated into four expression types. Type-I parasites express comparable levels of both trans-sialidase and sialidase activities (Endotrypanum species and Trypa- nosoma lewisi). Type-I1 parasites express predominantly trans-sialidase activity (Trypanosoma cruzi and Trypanosoma conorhini).Type-I11parasites express sialidase activity exclusively (Trypanosoma rangeli and Trypanosoma leeuwenhoeki). Type-IV parasites do not express either activity (Leish- mania species and Trypanoplasma borreli). The measurement of trans-sialidase and sialidase activi- ties thus permits the differentiation of parasites frequently found in the same insect vectors that are difficult to distinguish, such as T. cruzi and 7: rangeli, or in the same sylvatic vertebrate and invertebrate hosts, such as and Endotrypanum.

Trans-sialidases are a class of recently described cell-sur- of invasion of mammalian cells [3, 41, in defense against face-exposed trans-glycosidases that differ from the Golgi- complement attack [9], in the intracellular fate and survival associated sialyltransferases in that they catalyze the transfer of the parasite (reviewed in [lo-141). of N-acylneuraminate (sialic acid) residues from substrates Currently, it is difficult to discriminate between patho- other than cytidine monophosphate (CMP)-sialic acid to gly- genic and non-pathogenic trypanosomatids. Striking simi- coproteins, glycolipids and polysaccharides. Like all trans- larities at the morphological, molecular and biological levels glycosidases, the trans-sialidases also exhibit hydrolase (i.e. exist between many trypanosomatids isolated from sylvatic sialidaseheuraminidase) activity in the absence of suitable insects and/or vertebrate reservoir hosts that make the identi- acceptors. fication of the medically important parasites demanding (re- Trans-sialidase activity was discovered in the protozoan viewed in [15, 161). Moreover, the geographical distributions parasite Tqpanosoma cruzi (order , family of the different vectors and vertebrate reservoirs overlap. For Trypanosomatidae) [ 1- 51, the agent of Chagas' disease in example, the neotropical tree sloths (Edentata ; genera the American continent, and shortly after in their African Choleopus and Brudypus), which are reservoirs of at least counterparts Trypanosoma brucei ssp. [6, 71, the causative six Leishmania species pathogenic for humans (reviewed in organisms of sleeping sickness in humans and of nagana in [17]), are hosts of Endotrypanum (E. monterogeii and E. domestic mammals. Trans-sialidases are, however, not re- schaudinni; reviewed in [18]) and two Trypanosoma species stricted to trypanosomes as they also occur in the non-patho- (7: rangeli [19] and T. leeuwenhoeki [20]), none of which are genic trypanosomatids of the genus Endotrypanum [8], and pathogenic for humans. These parasites species have also in the pathogenic fungus Pneumocystis carinii (L. Trimble, been isolated from recently fed anthropophilic sandflies N. Pavia & M. E. A. Pereira, unpublished results). Although ( spp.) [ 19- 241. unequivocal evidence for the specific biological roles of the Endotrypanum parasites are unique among the Kineto- trans-sialidases in these parasite systems is lacking, the T. plastida in that they infect erythrocytes of the vertebrate host cruzi trans-sialidase is believed to be involved in the process [18], but these forms are rare in naturally or experimentally infected sloths. Diagnosis usually relies on the examination Correspondence to V. Nussenzweig, Michael Heidelberger Divi- of parasites from cultures or from sandflies, and those forms sion of Immunology, Department of Pathology, New York Univer- of the parasite are morphologically indistinguishable from sity, Medical Center, MSB Room 131, New York, NY 10016, USA Fax: +I212 263 8179. the promastigotes of Leishmania [25,261. Similarly, 7: cruzi Abbreviations. MeUmb-NeuAc, 2'(4-methylumbelliferyl) -D-N- and 7: rangeli can be found in the same vertebrate and insect acetylneuraminic acid. hosts [19, 27, 281. Although the blood-stage forms of T. Enzymes. Sialidase (EC 3.2.1 .IS); trans-sialidase. rangeli differ morphologically, they are exceedingly rare in 334 the circulation in the large majority of the infected popula- Enzymic activities tions. In contrast, the parasites obtained from the gut of the Sialidase activity was assayed by the hydrolysis of the vectors and from culture are not easily distinguishable solely fluorogenic substrate 2'(4-methylumbelliferyl)-a-~-N-acetyl- on the basis of morphology. neuraminic acid (MeUnib-NeuAc, Sigma) followed by quan- Even though several methods have been proposed to tification of the fluorescence emitted by methyl-umbellifer- discriminate these trypanosomatids [29 -401, the general one, with excitation at 350nm and reading emission at concensus is that they are cumbersome and not satisfactory, 460 nm in the Titertek FluorosKan 11. The final reaction mix- and that the definitive identification can be only attained by tures were 10 pl lysate (regularly containing 10 pg protein) recombinant DNA procedures [41-451. Here, we show that and 40 pl 1X substrate cocktail [0.2 mM MeUmb-NeuAc, the measurement of sialidase and trans-sialidase activities 0.005 % ultrapure bovine serum albumin (Boehringer Mann- also permits rapid diagnosis of culture forms of trypanoso- heim Corporation), in 100 mM sodium acetate, pH 7.01 or matids of medical importance. 40 pl supernatant and 10 pl 5X substrate cocktail. Maximal- activity values were achieved after an overnight incubation in the dark, at room temperature. The overnight incubation MATERIALS AND METHODS did not increase the blank, background or noise values, which Parasites never exceeded a row value of 70 arbitrary fluorescence units. The blank row values were subtracted from the sample Identification of the isolates, their geographic origin row values before normalization and calculation of the en- and the source of the stocks used in this study, are given in zymic activity. The reaction conditions were determined ex- Table 1. Unless otherwise indicated, parasites were grown at perimentally to provide maximal activity values (i.e. highest 25 "C in Schneider's Drosophila medium [46] supplemented sample versus blank ratios.) We have observed that the ratios with 20% heat-inactivated fetal bovine serum. T. cruzi and of sialidase/trans-sialidase activities are maintained indepen- ir: rangeli were cultured as described previously [47]. Trypa- dently of the time length (45 min versus 16 h) and/or temper- nosoma lewisi was grown in Sprague-Dawley weaning rats ature (22°C versus 4°C) of incubation. The reactions were that received a 0.5-ml inoculum from a frozen sample (pro- set up such that the measurements were maintained linear vided by J. Bienen and A. Clarkson, Jr, NYU Medical Center, with a dynamic range of 1- 1000 pmol standard methyl-um- USA) by intraperitoneal injection. Bloodstream fonns were belliferone and, of 0.5-100 pg protein lysate added. To en- isolated from freshly drawn blood (4 days after infection) by sure working under the linear range, we tested only 10 pg centrifugation through a Ficol1:Paque (Pharmacia) cushion protein. As monitored with standard solutions of methyl-um- layer, as recommended by the manufacturer for the in vitro bellyferone, one fluorescence unit corresponded to one pmol isolation of lymphocytes. Parasites (50%) were recovered methyl-umbellyferone. One unit sialidase activity is the from the interphase (lymphocyte layer) or the Ficoll-Paque amount of enzyme catalyzing the hydrolysis of 1 pmol phase (45%). For the isolation of the contaminant 7: cruzi MeUmb-NeuAc/min (1 U = 1 pmol . min-'). strain in the 7: rangeli San Agustin strain, four two-month- Trans-sialidase activity was determined by the transfer of old Swiss-background nude mice (nu/nu, Taconic) were inoc- sialyl residues from 3'-sialyllactose (donor/substrate) to [D- ulated intraperitoneally with 5 X lo7 parasites obtained from glucose-l-'4C]lactose (acceptor/substrate), in a 5O-pl reaction in vitro cultures. Blood samples were collected every two mixture containing 100 mM sodium acetate, pH 7.0, 50 nmol days and screened fresh for the presence of parasites. Blood sialyllactose and 370 pmol (54 Cdmol) radioactive lactose. smears were stained with Giemsa. Infection of LLC-MK, The mixture was incubated at room temperature for 45 min. cells was carried out as described by Schenkman et al. [47]. The reaction was then terminated by the addition of 1 ml water and loaded onto a I-ml QAE-Sephadex A50 column Sample preparation (Pharmacia LKB) equilibrated in water. The radioactive product (sialyl-[D-glucose-l-'4C]lactose) was eluted with Cells (1.5 ml 1X107-1X108 cells ml-l, from 5-8-day- 1 ml 1 M ammonium formate, and quantified by liquid scin- old cultures) were harvested in Eppendorf tubes by centrifu- tillation counting. One unit trans-sialidase activity is the gation at 1360Xg for 15 min at 4°C and washed once with amount of enzyme catalyzing the transfer of 1 pmol sialic ice-cold phosphate-buffered saline (0.15 M NaC1, 0.01 M so- acid from sialyllactose to radiolabelled-lactose/min. dium phosphate, pH 7.4) before lysis. The corresponding cul- ture supernatant was transferred to a fresh tube and cleared by centrifugation at 13000Xg for 30min at 4°C. The pel- DNA isolation and PCR amplification leted cells were suspended in lysis buffer (100 mM sodium Genomic DNA was isolated using the TELT mini-prep acetate, pH 7.0, containing 1.5% Nonidet P-40, 0.1 mM N- method [49]. Diagnostic PCR amplification of the T cruzi tosyl-L-lysine chloromethane, 0.1 mM phenylmethane- species-specific, repetitive, non-transcribed sequence SRE-1, sulfonyl fluoride and leupeptin at 25 pg ml-'), at 1X lo8cells was carried out as reported by Novak et al. [45]. ml-', by vigorous vortexing followed by a 60-min incubation on ice. Lysates were cleared by centrifugation at 13000Xg Statistical analysis for 30min at 4°C. Protein content was determined by the fluorescamine method, as described by Udenfriend et al. The differences in enzyme activity ratios between the [48j, using excitation at 350nm and reading emission at stocks were tested for statistical significance (P< 0.01) by 460 nm in a Titertek FluorosKan I1 (Flow Laboratories Inc., the Student's t-test (two-tailed) assuming equal variances. McLean, VA, USA). Peptides and polypeptides are known to yield maximum fluorescence near pH 7 .O, whereas for other reactive primary amines (amino acids, polyamines, etc.) fluo- RESULTS AND DISCUSSION rescence is generally maximal at pH 9.0 with little fluores- We measured and compared the levels of sialidase and cence at pH 7.0. trans-sialidase activities from both cell extracts and culture 335

Table 1. Origin and identification of the kinetoplastid reference strains, stocks and isolates employed in this study. Designations. M = Mammalia; BRA = Bradypus; CHO = Choloepus; HOM = Homo sapiens; CEB = Cebus apella; I = Insecta: SHA = Lutzornyia shanonni s.1. Data were acquired from the culture collections of J. J. Shaw, Instituto Evandro Chagas/Wellcome Parasitology Unit, BelCm, PA, Brazil, 'M. P. Deane and A. M. R. Franco and G. Grimaldi Jr., Instituto Oswaldo Cruz/F'IOCRUZ, Rio de Janeiro, RJ, Brazil, American Type Culture Collection, Rockville, MD. USA, A. H. C. S. Lopes, Instituto de MicrobiologiaLJFRJ, Rio de Janeiro, RJ, Brazil, ' T. V. Barrett, Instituto Nacional de Pesquisa da Amazonia, Manaus, AM, Brazil, ' E. S. Garcia, Instituto Oswaldo Cruz/FIOCRUZ, Rio de Janeiro, RJ, Brazil, * P. Michels, ILP-TROP, Belgium, A. Clarkson, Jr., New York University, New York, USA. Stock identification of the Brazilian isolates was established by isoenzyme, monoclonal antibodies and molecular karyotype analyses (Franco, A. M. R., Momen, H. and Grimaldi, Jr. G., unpublished results).

Stock Designation Species Locale Source

Reference strains

TO1 MBRA/CO/OO/BV388 a Trypanosorna leeuwenhoeki Colombia 1 TO2 MCHO/CO/OO/CH250' 1: leeuwenhoeki Colombia 1 TO5 MHOM/BR/OO/F strain 7: cruzi Brazil 2 TO6 Providencia 7: conorhini Brazil 2 TO7 MPD 7: conorhini Brazil 2 TO8 M238 1: sp. (bat) Brazil 2 TO9 ILP-TROP Trypanoplasrna borreli - 8 T10 30085-Y 1: lewisi U.S.A. 9 T21 sc-1 1: rangeli Brazil 7 T22 SC-58 1: rangeli Brazil 2 T23 SC-72 I: rangeli Brazil 2 T24 SC-61 1: rangeli Brazil 2 T25 San Agustin 1: rangeli Brazil 2 T26 T25 subclone 7: cruzi U.S.A. - H01 IMEG/US/72/H.meg Herpetornonas rnegaseliae U.S.A. 2 LO2 MHOM/BR/75D44147 Leishmania guyanensis Brazil 3 LO3 MCEB/BR/84/M8408 L. shawi Brazil 3 E09 MBRAPA/O0/415P01 Endotrypanurn sp. Panama 1 Ell MCHO/CR/62/A-9 E. rnonterogeii Costa Rica 1 El 2 MCHO/BR/88/Ml1602 E. schaudinni Brazil 1 E49 MCHO/C0/68/473 Endotrypanurn sp. Colombia 4 Endotrypanu'rn stocks E08 ISHAIBR/80/IM217 E. schaudinni Brazil 2 El 0 MBRA/PA/81/1222P82 Endotrypanurn sp. Panama 1 El 3 MCHO/PA/79/GML-30 E. schaudinni Panama 1 El4 MCHO/BR/80/M6159 E. schaudinni Brazil 1 El5 MCHO/BR/79/M5725 E. schaudinni Brazil 1 E42 MCHOICR/OO/LV88 E. rnonterogeii Costa Rica 5 E43 MCHO/PA/OO/LV59 E. schaudinni Panama 5 E44 MCHO/CO/OO/Col E. schaudinni Colombia 5 E45 MCHO/PA/72/3130 Endotrypanurn sp. Panama 5 E46 MCHO/C0/68/473 Endotrypanurn sp. Colombia 5 E47 MCHO/BR/80/M6159 E. schaudinni Brazil 5 E48 MCHO/BR/OO/LV86 E. schaudinni Brazil 5 Brazilian isolates E01 MCHO/BR/89/R09627 Endotrypanurn sp. Rondonia 3 E02 MCHO/BR/89/R01635 Endotrypanurn sp. Rondonia 3 E03 MCHO/BR/89/RO1634 Endotrypanurn sp. Rondonia 3 E04 MCHO/BR/89/R01140e Endotrypanurn sp. Rondonia 3 E05 MCHO/BR/89/RO1602' Endotrypanurn sp. Rondonia 3 E06 MCHO/BR/89/R0147 1 Endotrypanurn sp. Rondonia 3 E07 MCHO/BR/89/RO1583 Endotrypanurn sp. Rondonia 3 El6 MCHO/BR/85/IM2260 Endotrypanurn sp. Para 6 El7 MCHO/BR/85/IM2382 Endotrypanurn sp. Rondonia 6 El 8 MCHO/BR/85/IM2384" Endotrypanurn sp. Rondonia 6 El9 MCHO/BR/85/IM2389 Endotrypanurn sp. Rondonia 6 E20 MCHO/BR/85/IM2404 Endotrypanurn sp. Amazonas 6 E21 MCHO/BR/89/IM3605 Endotrypanurn sp. Rondonia 6 E22 MCHO/BR/89/IM3606 Endotrypanurn sp. Rondonia 6 E29 ISHA/BR/80/IM1111 Endotrypanurn sp. Amazonas 6 E3 1 MCHO/BR/85/IM2259 Endotrypanurn sp. Para 6 E32 MCHO/BR/85/IM2380e Endotrypanurn sp. Rondonia 6 E33 MCHO/BR/85/IM2393 Endotrypanurn sp. Rondonia 6 E36 MCHO/BR/89/IM3603 Endotrypanurn sp. Rondonia 6

a Host was B. tridactylus; host was B. infuscatus; ' host was C. hoffmanni; host was C. didactylus; host was C. juruanus. 336 Table 2. Sialidase and trans-sialidase expression types in culture A forms of trypanosomatids. The activity ratios shown are from cell 1000 - lysates. Within each expression type, the differences between the ratios from lysates and supernatants are not statistically significant (t = P>0.1).

h 100 Type Activity ratios Parasite Q (sialidaseltrans- -m u sialidase) u) 0) 0 IA" 0.97 f 0.15 Endotrypanum schaudinni j 10 * Endotrypanurn spp. .-c Trypanosoma lewisi .-> B 2.27 5 0.86 E. monterogeii U E. schaudinni 91 Endotrypanum spp. I1 0.10 2 0.06 Trypanosoma cruzi T. conorhini pq Trypanosoma sp. (bat) 0.1 111 74.710.0 7: leeuwenhoeki Expression Types 49.210.1 7: leeuwenhoeki 180.3 2 18.0/0.0 T. rangeli

100- IV 0.010.0 Trypanoplasma borreli B Leishmania guyanensis I II IV L. shawi Herpetomonas megaseliae v as in [8] L. mexicana, L. donovani, L. major, L. chagasi, 0 L. amazonensis, H. samuelpessoae, Phytomonas davidi, Leptomonas seymouri, Crithidia fasciculata, C. deanei, Blastocrithidia culicis

a The differences between the ratios in A and B are statistically significant (t = P < 0.01). l Blood stages of the parasite (see Materials and Methods).

Expression Types Fig. 1. Levels of sialidase (SA) and trans-sialidase (TSA) enzyme trans-sialidase enzyme activity ratios shown in Table 2. Type activities in cell lysates (A) or culture supernatants (B) of the I is represented by 23 Endotrypanum stocks, which express kinetoplastids listed in Table 1. Shown is a distribution along the comparable levels of both activities. Two subgroups can be y-axis of the activity levels in the four (I-IV) parasite expression distinguished: (A) in which the activity ratios are 0.97 t 0.15 types. The y-axis is drawn in logarithmic scale. The relative enzymic (13 stocks), and (B) in which the ratios are 2.275036 (11 activity in each sample is expressed as pU mg protein-'. Each value stocks), Table 2. Note, in addition, that the absolute levels of represents the mean number of duplicates from one of three indepen- enzymic activities within each subgroup vary greatly (from dent experiments. Variations between duplicates and experiments 0.1 -164 pU . mg-'), Fig. 1. The reason(s) for these varia- were less than 5%. tions is unknown. Artifacts such as differences in the growth stage of the parasites or proteolytic degradation of the en- zyme during sample preparation are unlikely ; all Endotrypa- supernatants of 52 parasite stocks comprising the two sub- num extracts were prepared from 5-day-old cultures, and the orders of the kinetoplastida, Trypanosonzatina (family Try- ratios of the activities were remarkably constant, regardless panosomatidae; genera Leishmania, Endotrypanum, Trypa- of the incubation conditions (time length and temperature) of nosoma and Herpetomonas) and Bodonina (family Crypto- the assays. The marked differences in the levels of sialidase biidae; genus Trypanoplasma), Table 1. The stocks, which and trans-sialidase enzyme activities observed among the En- were originally isolated from different hosts (human, wild dotrypanum stocks may be genetically determined and per- and domestic animals or sandflies) and geographic areas, haps represent strain-specific traits of a species complex [18, were selected in order to represent the pathogenic and non- 521. The heterogeneity of Endotrypanum stocks was also pathogenic parasites that are morphologically indistinguish- noted by Franco et al. [36] who analyzed 27 of the 35 Endo- able, i.e. within the genus Trypanosoma, we examined both trypanum stocks used here by isoenzyme electrophoresis. A pathogenic (7: cruzi) and non-pathogenic (7: lewisi [50],7: comparison of the electrophoretic profiles with those from conorhini [Sl], 7: rangeli and is leeuwenhoeki) parasites. reference strains representing the genus revealed that the en- Fig. 1 depicts the levels of enzymic activities present in zymic loci were highly polymorphic. cell extracts (A) and culture supernatants (B). The stocks fall Type I1 includes is cruzi and 7: conorhini, which pre- into four expression types (I-IV), according to the sialidase/ dominantly express trans-sialidase activity. The ratio of siali- 337 daseltrans-sialidase activities in extracts or culture superna- An obvious point raised here that awaits further experi- tants of these parasites is 0.1 & 0.06. mentation is the biological significance of the trans-sialidase Type 111, represented by 2: rangeli and 7: leeuwenhoeki activity in the culture forms of 7: conorhini and 7: leeuwen- stocks, is characterized by high levels of sialidase activity hoeki for which neither an intracellular lifestyle, nor a patho- and no, or very little, trans-sialidase activity. In stock T02, genic activity in the mammalian hosts have been described. we consistently obtained 0.1 pU mg-' trans-sialidase, a value Similarly, both sialidase and trans-sialidase activities (1 : 1 which falls close to the sensitivity limit of the assay. ratio) are expressed in the blood stages of I: lewisi, a rodent Type-IV parasites do not express either activity. Included parasite that does not enter hosts cells. in this category are the Leishmania species, Herpetomonas Another important issue to be resolved is the sialylation megaseliae, Trypanoplasma borreli and 11 stocks that were states of the membrane glycoconjugates of Type-I parasites. originally identified as Endotrypanum. These 11 stocks differ It is conceivable that there is a dynamic equilibrium between from the Endotrypanum reference strains by morphological, the sialylated and desialylated states. In contrast, some of the karyotypic, isoenzymic and in vitro growth rate criteria ([36], surface components may not be accessible to the sialidase and Franco et al., unpublished observations). Moreover, two activity (if this is a separate enzyme) and they remain mostly of these stocks (E46 and E47) came from reference strains sialylated in the presence of suitable donors. that expressed both activities (E49 and E14, respectively). Lastly, the lack of sialidasehans-sialidase enzyme activi- These observations argue against the authenticity of the non- ties in the epimastigote forms of the cryptobiid 7: borreli expressing stocks as belonging to the genus Endotrypanum, poses a challenging problem on the origin(s) of the trypano- and suggest that the original stocks were contaminated or soma1 enzymes. Current phylogenetic trees inferred from mislabelled. comparisons of nuclear small-subunit rRNA sequences from In short, sialidase and trans-sialidase activities were de- several kinetoplastids [54-58] indicate that 7: borreli tected in all reference Endotrypanum strains (Type I), inde- branches off early from the trypanosomatid lineage. Hence, pendently of the reservoir host or the geographic area of iso- the occurrence of the sialidase and trans-sialidase genetic in- lation, while promastigotes of all Leishmania species exam- formation in the trypanosomes may have taken place after ined ([S], and this report), including L. guyanensis (which the separation of these lineages. Alternatively, the ancestor shares with Endotrypanum spp. both the sloth and sandfly of 7: borreli may have lost the information during evolution. hosts), lack both activities (Type IV). Further comparative studies will be needed to address the We have also extended the previous observations that the antiquity of these enzymes and, in particular, whether or not 7: rangeli sialidase(s) lacks trans-sialidase activity [40, 531, they are present in the bodonids and the deeper rooted neigh- while the I: cruzi enzyme exhibits both sialidase and trans- bours, the euglenoid . sialidase activities [5].We assayed extracts of epimastigotes from five different strains of 7: rangeli and found that they We thank the providers of the various strains and stocks used in do not express detectable trans-sialidase activity. this study, listed in Table 1. We are grateful to them for their gener- The discriminatory power of the enzymic assays used ous donations. This work was supported by grants from the John D. here is best illustrated by the analysis of I: rangeli San Agus- and Catherine T. MacArthur Foundation and NIH (AI32966). We thank S. Paul and Chien-Kuo Lee for valuable suggestions on the tin strain (stock T25, Table 1). We found that stock T26 of manuscript. this strain contained high levels of trans-sialidase activity and, suspecting contamination with 2: cruzi, we performed several additional tests. Injection into nude mice yielded high REFERENCES levels of parasitemia, and the bloodstream trypomastigotes were morphologically indistinguishable from 7: cruzi. This I: 1. Previato, J. O., Andrade, A. F., Pessolani, M. C. & Mendoqa- rangeli stock infected LLC-MK cells in vitro, and multiplied Previato, L. (1985) Incorporation of sialic acid into Trypano- soma cruzi macromolecules. A proposal for a new metabolic intracellularly as amastigotes. An axenic culture of the exit- route, Mol. Biochem. Parasitol. 16, 85-96. ing trypomastigotes at 28 "C did not contain the pleomorphic 2. Zingales, B., Carniol, C., de Lederkremer, R. M. & Colli, W. parasite population typical of 7: rangeli stocks T21 -T24, (1987) Direct sialic acid transfer from a protein donor to gly- but was morphologically indistinguishable from 2: cruzi. The colipids of trypomastigote forms of Tqpanosoma cruzi, Mol. bloodstream forms, the tissue-culture trypomastigotes and Biochem. Parasitol. 26, 135 - 144. the epimastigote thus generated from strain T26, all ex- 3. Piras, M. M., Henriquez, D. & Piras, R. (1987) The effect of pressed trans-sialidase activity. To confirm independently fetuin and other sialoglycoproteins on the in vitro penetration that these forms were 7: cruzi, we carried out a differential of Trypanosoma cruzi trypomastigotes into fibroblastic cells, PCR analysis. 7: cruzi DNA was detected by amplification Mol. Biochem. Parasitol. 22, 135-143. 4. Schenkman, S., Jiang. M.-S., Hart, G. W. & Nussenzweig, V. of the 7: cruzi-specific ribosomal spacer repetitive element (1991) A novel cell surface trans-sialidase of Trypanosoma SRE-1 [45] which is absent in the genome of 7: rangeli (not cruzi generates a stage-specific epitope required for invasion shown). of mammalian cells, Cell 65, 1117-1125. Our results are in good agreement with those reported by 5. Schenkman, S., Pontes de Carvalho, L. & Nussenzweig, V. Schottelius 1391 and Reuter et al. [40], who also distin- (1992) Trypanosoma cruzi trans-sialidase and neuraminidase guished I: rangeli from 7: cruzi epimastigotes using a siali- activities can be mediated by the same enzymes, J. Exp. Med. dase fluorescence test on culture supernatants. However, it 175, 567-575. should be noted that, unless cloning of the parasites is carried 6. Pontes de Carvalho, L. C., Tomlinson, S., Vandekerckhove, F., out, the fluorescence test cannot discriminate cruzi from Bienen, E., Clarkson, A. B., Jiang, M. S., Hart, G. M. & I: Nussenzweig, V. (1993) Characterization of a novel trans-sia- I: rangeli in mixed infections or contaminated cultures, (i.e. lidase of Trypanosoma brucei procyclic trypomastigotes and high levels of sialidase activity reflect either the presence of identification of procyclin as the main sialic acid acceptor, J. 7: rangeli exclusively or a mixed infection with 7: cruzi). The Exp. Med. 177,465-474. measurement of trans-sialidase activity, as illustrated above, 7. Engstler, M., Reuter, G. & Shauer, R. (1993) The developmen- resolves this diagnostic problem. tally regulated trans-sialidase from Trypanosoma brucei sialy- 338

lates the procyclic acidic repetitive protein, Mol. Biochem. (Lumsden, W. H. R. & Evans, D. A,, eds) vol. 1, pp. 327- Parasitol. 61, 1-14, 493, Academic Press, London. 8. Medina-Acosta, E., Paul, S., Tomlinson, S. & Pontes-de-Car- 28. Steindel, M., Carvalho Pinto, J. C., Toma, H. K., Mangia, R. H. valho, L. (1994) Combined occurrence of trypanosomal siali- R., Ribeiro-Rodrigues, R. & Romanha, A. J. (1991) Trypano- dase/trans-sialidase activities and leishmanial metalloprotein- soma rangeli (Tejera, 1920) isolated from a sylvatic rodent ase gene homologues in Endotrypanum sp., Mol. Biochem. (Echimys dasythrir) in Santa Catarina Island, Santa Catarina Parasitol.64, 273 - 282. State: First report of this trypanosome in the Southern Brazil, 9. Tomlinson, S., Pontes-de-Carvalho, L. C., Vanderherkhove, F. & Mem. Inst. Oswaldo Cruz 86, 73-79. Nussenzweig, V. (1994) The role of sialic acid in the resis- 29. Schottelius, J. (1982) Differentiation between Trypanosoma tance of Trypanosome cruzi trypomastigotes to complement, cruzi and T rangeli by their different complement sensitivity, J. Immunol., in the press. Tropenmed. Parasitol. 33, 147- 150. 10. Cazzulo, J. J. & Frasch, A. C. C. (1992) SAPMtrans-sialidase 30. Schottelius, J. & Muller, V. (1984) Interspecific differentiation and cruzipain : two antigens from Trypanosoma cruzi contain of Trypanosoma cruzi, Trypanosoma conorhini and Trypano- immunodominant but enzymatically inactive domains, FASEB soma rangeli by lectins in combinations complement lysis, J. 6, 3259-3264. Acta Trop. 41, 29-38. 11. Colli, W. (1 993) Trans-sialidase: a unique enzyme activity dis- 31. Miranda Santos de. I. K. F. & Pereira, M. E. A. (1984) Lectin covered in the Drotozoan Trypanosoma__ cruzi, FASEB J. 7, discriminate between pathogenic and nonpathogenic South 1257- 1264. American trypanosomes, Am. J. Trop. Med. Hyg. 33, 839- 12. Schenkman, S. & Eichinger, D. (1993) Trypanosoma cruzi 844. trans-sialidase and cell invasion, Parasitol. Today 9, 218- 32. Anthony, R. L., Williams, K. M., Sacci, J. B. & Rubin, D. C. 222. (1985) Subcellular and taxonomic specificity of monoclonal 13. Engstler, M. & Schauer, R. (1993) Sialidases from African try- antibodies to New World Leishmania, Am. J. Trop. Med. Hyg. panosomes, Parasitol. Today 9, 222-225. 34, 1085-1094. 14. Schenkman, S., Eichinger, D., Pereira, M. E. A. & Nussen- 33. Hudson, L., Guhl, F., Marinkelle, C. J. & Rodriguez, J. (1987) zweig, V. (1994) Structural and functional properties of Try- Use of monoclonal antibodies for the differential detection of panosoma trans-sialidase, Annu. Rev. Microbiol. 48, 499- Trypanosoma cruzi and Trypanosoma rangeli in epidemiolog- 523. ical studies and xenodiagnosis, Acta Trop. 44, 387-392. 15. Zeledbn, R. (1974) Epidemiology, modes of transmission and 34. Lopes A. H. & McMahon-Pratt, D. (1989) Monoclonal antibod- reservoir host of Chagas’ disease, in Trypanosomiasis and Leishmaniasis, Ciba Foundation Symposium 20, new series, ies specific for members of the genus Endotrypanum, J. Pro- pp. 51 - 85. Associated Scientific Publishers, Amsterdam. tozool. 36, 354-361. 16. Molyneux, D. H. & Ashford, R. W. (1983) The biology of Try- 35. Ebert, F. (1986) Isoenzymes of Trypanosoma rangeli stocks and panosoma and Leishmania, parasites of man and domestic their relation to other trypanosomes transmitted by triatomine animals, Taylor & Francis Ltd., London. bugs, Trop. Med. Parasitol. 37, 251 -254. 17. Grimaldi, Jr., G. & Tesh, R. B. (1993) Leishmaniases of the 36. Franco, A. M. R., Moreira, C. F. S., Camera, C., Carvalho-Paes, New World: Current concepts and implications for future re- L. E., Momen, H., Deane, M. P., Grimaldi, G. Jr (1993) Char- search, Clin. Microbiol. Rev. 6, 230--250. acterization of the genus Endotrypanum by isoenzymatic 18. Shaw, J. J. (1992) Endotrypanum, a unique inhaerythrocytic analysis, Mern. Inst. Oswaldo Cruz 88, 298. of New World tree sloths. An evolutionary link or 37. Pacheco, R. S., Thomaz, N. & Momen, H. (1990) kDNA cross- an evolutionary backwater?, CiCncia e Cultura 44, 107-116. hybridization between Endotrypanum and peripylarian Leish- 19. Miles, M. A., Arias, J. R., Valente, S. A., Naiff, R. D., de Souza, mania, Trans. Roy. Soc. Trop. Med. Hyg. 84, 531. A. A., Pbvoa, M. M., Lima, J. A. & Cedillos, R. A. (1983) 38. Vallejo, G. A., Chiari, E., Macedo, A. M. & Pena, S. D. J. Vertebrate hosts and vectors of Trypanosoma rangeli in the (1993) A simple laboratory method for distinguishing be- Amazon Basin of Brazil, Am. J. Trop. Med. Hyg. 32, 1251 - tween Trypanosoma cruzi and Trypanosoma rangeli, Trans. 1259. Roy. Soc. Trop. Med. Hyg. 87, 165-166. 20. Travi, B. L., Zea, A. & D’Alessandro, A. (1989) Trypanosoma 39. Schottelius, J. (1987) Neuraminidase fluorescence test for the (Herpetosoma) leeuwenhoeki in Choloepus ho&ani and Di- differentiation of Trypanosoma cruzi and Trypanosoma delphis marsupialis of the Pacific Coast of Colombia, J. Para- rangeli, Trop. Med. Parasitol. 38, 323 -327. sitol. 75, 218-224. 40. Reuter, G, Schauer, R., Prioli, R. & Pereira, M. E. A, (1987) 21. Shaw, J. J. (1963) A possible vector of Endotrypanum schau- Isolation and properties of a sialidase from Trypanosoma dinni of sloth, Choleopus hoffmanni, in Panama, Nature 201, rangeli, Glycoconjugate J. 4, 339-348. 417-418. 41. Greig, S. R., Akinsehinwa, F. A., Ashall, F., Lainson, R., Shaw, 22. Christensen, H. A. & Herrer, A. (1976) Neotropical sand flies J. J., Miles, M. A. & Barker, D. C. (1989) The feasibility of (Diptera: Psychodidae), invertebrate hosts of Endotrypanurn discriminating between Leishmania and Endotrypanum using schaudinni (Kinetoplastida: Trypanosomatidae), J. Med. En- total parasite DNA probes, Trans. Roy. Soc. Trop. Med. Hyg. tomol. 13, 299-303. 83, 196-197. 23. Christensen, H. A. & de Vasquez, A. M. (1982) The tree- 42. Gonqanves, A. M., Nehme, N. S., Saravia, N., Segura, I. & buttress biotope: a pathobiocenose of Leishmania brazitiensis, Morel, C. M. (1991) Schizodeme analysis with the restriction Am. J. Trop. Med. Hyg. 31, 243-251. endonuclease RSA I differentiates between Trypanosoma 24. Arias, J. R., Miles, M. A., Naiff, R. D., Pbvoa, M. M., de Frei- tas, R. A,, Biancardi, C. B. & Castellon, E. G. (1985) Flagel- rangeli and Trypanosoma cruzi, Mem. Inst. Oswaldo Cruz 86, 477 -478. late infections of Brazilian sandflies (Diptera:Psychodidae) : isolation in vitro and biochemical identification of Endotrypa- 43. Fernandes, O., Degrave, W. & Campbell, D. A. (1993) The num and Leishmania, Am. J. Trop. Med. Hyg. 34, 1098-1108. mini-exon gene: a molecular marker for Endotrypanum 25. Croft, S., Chance, M. & Gardener, P. J. (1980) Ultrastructural Schaudinni, Parasitol. 107, 219-224. and biochemical characterization of stocks of Endotrypanum, 44. Murthy, V. K., Dibbern, K. M. & Campbell, D. A. (1992) PCR Ann. Trop. Med. Parasitol. 74, 585-589. amplification of mini-exon genes differentiates TrypanoSoma 26. Soares, M. M., Lopes, A. H. C. S. & de Sousa, W. (1990) Ultra- cruzi from Trypanosoma rangeli, Mol. Cell, Probes 6, 237- structural and steriological analysis of trypanosomatids of the 243. genus Endotrypanurn, Mern. Inst. Oswaldo Cruz 86, 175- 45. Novak, E. M., de Mello, M. P., Gomes, H. B. M., Galindo, I., 180. Guevara, P., Ramirez, J. L. & Franco da Silva, J. (1993) Re- 27. D’Alessandro, A. (1976) Biology of Trypanosoma (Herpeto- petitive sequences in the ribosomal intergenic spacer of Try- soma) rangeli Tejera (1920) in Biology of Kinetoplastida, panosoma cruzi, Mol. Biochem. Parasitol. 60, 273-280. 339

46. Hendricks, L. D., Wood, D. E. & Hadjuk, M. E. (1978) Haemo- somal DNAs in the trypanosomatid genus Endotrypanum, flagellates : commercially available liquid media for rapid cul- Mol. Biochem. Parasitol. 40, 151- 162. tivation, Parasitol. 74, 309-316. 53. Pontes de Carvalho, L. C., Tomlinson, S. & Nussenzweig, V. 47. Schenkman, S., Robbins, E. S. & Nussenzweig, V. (1991) At- (1 993) Trypanosoma rangeli sialidase lacks trans-sialidase ac- tachment of Trypanosoma cruzi to mammalian cells requires tivity, Mol. Biochem. Parasitol. 62, 19-26. parasite energy, and invasion can be independent of the target 54. Cavalier-Smith. T. (1993) Kingdom protozoa and its 18 phyla, cell cytoskeleton. Infi Immun. 59, 645-654. Microbiol. Rev. 57, 953 -994. 48. Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber, 55. Fernandes, A. P., Nelson, K. & Beverly, S. M. (1993) Evolution W. & Weigele, M. (1972) Fluorescarnine: a reagent for assay of nuclear ribosomal RNAs in kinetoplastid protozoa: per- of amino acids, peptides, proteins, and primary amines in the spectives on the age and origins of parasitism, Proc. Natl picomole range, Science 178, 871 - 872. Acad. Sci. USA 90, 11608-11612. 49. Medina-Acosta, E. & Cross, G. A. M. (1993) Rapid isolation of 56. Landweber, L. F. & Gilbert, W. (1994) Phylogenetic analysis of DNA from trypanosomatid protozoa using a simple “mini- RNA editing: A primitive genetic phenomenon, Proc. Natl prep” procedure, Mol. Biochem. Parasitol. 59, 327-330. Acad. Sci. USA 91, 918-921. 50. Brown, W. H. (1914) A note on the pathogenicity of Trypano- 57. Maslov, D. A., Avila, H. A., Lake, J. A. & Simpson, L. (1994) soma lewisi, J. Exp. Med. 19, 406-410. Evolution of RNA editing in kinetoplastid protozoa, Nature 51. Deane, M. P. & Kirchner, E. (1963) Life cycle of Trypanosoma 368, 345-348. conorhini. Influence of temperature and other factors on 58. Briones, M. R. S., Egima, C. M., Eichinger, D. & Schenkman, growth and morphogenesis, J. Protozool. 10, 391 -399. S. (1 994) Trans-sialidase genes expressed in mammalian 52. Lopes, A. H., Iovannisci, D., Petrillo-Peixoto, M., McMahon- forms of Trypanosoma cruzi evolved from ancestor genes ex- Pratt, D. & Beverley, S, M. (1990) Evolution of nuclear DNA pressed in insect forms of the parasite, J. Mol. Evolution, in and the occurrence of sequences related to new small chromo- the press.

Supplementary Material. Trans-sialidase and sialidase activities discriminate between morphologically indistinguishable trypanosomatids. The raw data used to generate Fig. 1 are available, upon request, from the Editorial Office. A total of two pages are available.