mental stages: amastigote, epimastigote, and trypomasti- gote. The infective trypomastigote form has been shown to express higher levels of neuraminidase ...
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Vol. 59, No. 1
0019-9567/91/010464-03$02.00/0 Copyright © 1991, American Society for Microbiology
Differential Expression of Trypanosoma cruzi Neuraminidase in Intra- and Extracellular Trypomastigotes I.
A. ROSENBERG,t R. P. PRIOLI, J. S. MEJIA, AND M. E. A. PEREIRA*
New England Medical Center Hospitals, Department of Geographic Medicine and Infectious Diseases, 750 Washington Street, P. 0. Box 041, Boston, Massachusetts 02111 Received 27 August 1990/Accepted 2 November 1990
The developmentally regulated expression of Trypanosoma cruzi neuraminidase in culture cells was monitored by immunofluorescence with a monoclonal antibody (TCN-2) against the enzyme. The results showed that TCN-2 reacted with all intracellular trypomastigote forms (NA') but not with amastigotes. Immunoprecipitation of radiolabeled neuraminidase confirmed TCN-2 reactivity with trypomastigotes and the specificity of the antibody binding. During exiting from the host cells, all trypomastigotes were still NA+. However, when free in the extracellular environment, the relative proportion of NA' parasites declined from 100% to about 20%, thereby establishing a subpopulation of trypomastigotes which did not express enzyme (NA-). The expression of neuraminidase in all intracellular trypomastigotes and in only a subpopulation of the extracellular counterpart suggests that the enzyme may play a role in parasite exiting from infected cells.
Trypanosoma cruzi is the causative agent of Chagas' disease, and its life cycle includes three different developmental stages: amastigote, epimastigote, and trypomastigote. The infective trypomastigote form has been shown to express higher levels of neuraminidase activity than epimastigotes, and amastigotes do not have detectable enzyme activity (4). The role neuraminidase may play in the T. cruzi infection is not fully understood, although a growing body of evidence suggests the enzyme may be important during host-parasite interaction. For instance, trypomastigotes have been shown to desialylate endothelial and myocardial cells in vitro (3), and the degree of erythrocyte desialylation has been correlated with the levels of parasitemia in mice (3) and humans (9). Furthermore, neuraminidase activity has been inversely correlated with virulence of T. cruzi strains (5), and inhibitors of enzyme activity promoted trypomastigote infection of host cells in vitro (1, 7). In addition, immunofluorescence analysis of tissue culture trypomastigotes with polyclonal (1) or monoclonal (7) antibodies to T. cruzi neuraminidase revealed a parasite subset (20 to 30%) that expresses neuraminidase (NA') and another one (70 to 80%) that does not (NA-). The two subpopulations differ in their abilities to infect host cells, and under defined in vitro conditions, NA- trypomastigotes were more infective than either NA' or the total trypomastigote population (1). In light of the possible influence the neuraminidase may be exerting on the infection process, it would be of interest to determine the fate of the enzyme during parasite development in mammalian cells. Trypomastigotes of the clone Silvio X-10/4 of T. cruzi (6) were used to infect Vero cells as described previously (7). To follow the transformation of amastigotes into trypomastigotes, Vero cells were plated in four-chamber tissue culture slides (Lab-Tek, Naperville, Ill.), grown at 37°C in a 5% CO2 atmosphere in RPMI supplemented with 5% NU serum (Collaborative Research, Bedford, Mass.) to subconfluency, and infected with trypomastigotes at a ratio of 10 parasites to
1 Vero cell. After a 2-h infection period, unattached parasites were removed by being washed with RPMI, and the monolayers were incubated at 37°C in the presence of RPMI and 5% NU serum for up to 5 days. Indirect immunofluorescence assay was used to monitor the expression of neuraminidase as follows. Methanol-fixed monolayers of infected Vero cells were incubated for 30 min with a 1:50 dilution of normal goat serum (Sigma, St. Louis, Mo.) to block sites of nonspecific binding. After being rinsed three times with phosphate-buffered saline (PBS), the slides were exposed to 5 jig of TCN-2 or an isotype-matched anti-arsonate antibody (8) per ml for 60 min at room temperature, washed three times in PBS, and incubated for 30 min with fluorescein-conjugated goat anti-mouse immunoglobulin G (50 jig/ml). After being washed with PBS the slides were mounted with a mounting fluid (90% glycerol, 10 mM K2HPO4, 1 mg of p-phenylenediamine per ml), covered by a glass coverslip, and sealed with nail polish. Extracellular trypomastigotes were metabolically labeled with [35S]methionine as previously described (7). Intracellular parasites were metabolically labeled as follows. Monolayers of infected Vero cells containing mainly amastigotes (60 to 70 h postinfection) or trypomastigotes (80 to 90 h postinfection) were washed three times with methionine-free RPMI to remove extracellular parasites. The parasitized cells were then incubated with 250 p.Ci of [35S]methionine (ICN, Irvine, Calif.) in 5 ml of methionine-free RPMI for 1 h at 37°C with agitation on a rocker platform. After the monolayers were washed as described above, the cells were scraped, lysed in 1 ml of 1% Triton X-100 for 1 h at 4°C, and centrifuged in a microcentrifuge for 15 min. Labeled enzyme from intra- and extracellular parasites was precipitated with an immunocomplex prepared with TCN-2 and sheep antimouse immunoglobulin G as previously described (7). Immunocomplexes were electrophoresed on a sodium dodecyl sulfate (SDS)-7.5% polyacrylamide gel and transferred to nitrocellulose paper. The dried paper was processed for autoradiography by exposing the dried membrane to Kodak X-Omat-AR film for 12 to 24 h in a Cronex Quanta III screen film holder (Dupont). Indirect immunofluorescence assay was performed at various times during the course of infection of host cells by T.
Corresponding author. t Present address: Cancer Center, Massachusetts General Hospital East, Charlestown, MA 02129. *
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FIG. 1. Immunofluorescence staining with TCN-2 of T. cruzi-infected Vero cells at different times following infection. Phase contrast picture (A) shows intracellular amastigotes which were not stained by TCN-2 (B). Neuraminidase was detected punctated in the newly transformed trypomastigotes at about 76 h postinfection (C) and throughout the parasite at around 80 to 90 h postinfection (D, phase contrast; E, immunofluorescence of the same cell). When trypomastigotes were released during rupture of the Vero cell (F), they were still all reactive with TCN-2 (G). In the extracellular milieu (H), the proportion of NA' trypomastigotes declined to about 20% (I). Photographs were taken with a Nikon epifluorescence system for Optiphot and Labophot with an HFX-II automatic exposure system. Magnification, x 360.
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B
C
D
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FIG. 2. Immunoprecipitation of 35S-labeled neuraminidase from intra- and extracellular parasites. Radiolabeled enzymes from intracellular (lane A) and extracellular (lane B) trypomastigote and amastigote (lane C) lysates were immunoprecipitated with TCN-2 as described in the text. Lane D, Immunoprecipitation of radiolabeled intracellular neuraminidase with an immunoglobulin G2b anti-arsonate monoclonal antibody (8). Arrows indicate molecular weight in thousands.
cruzi. No reaction was observed with cells infected for less than 70 h because these cells contained only amastigotes (Fig. 1A and B). Between 72 and 76 h, TCN-2 was found to be reactive with newly transformed trypomastigotes; the reaction was in a subcellular compartment that appeared to be situated between the nucleus and the kinetoplast (Fig. 1C). Neuraminidase expression was enhanced in Vero cells that were infected for longer periods (Fig. 1D and E). It can be concluded that all intracellular trypomastigotes were reactive with TCN-2 and therefore are NA'. Trypomastigotes were still all NA' when they exited the Vero cells during cell burst, a finding readily observed at 90 to 100 h postinfection (Fig. 1F and G). In contrast, when released into the extracellular environment, freely swimming trypomastigotes contained only 20% NA' (Fig. 1H and I), in agreement with previous findings (1, 7). The proportions of NA' and NA- trypomastigotes in the extracellular milieu were constant at around 20 and 80%, respectively, whether swimming parasites were harvested after 1, 2, or 3 days after cell burst. The control anti-arsonate antibody did not react with trypomastigotes. In order to rule out the possibility that the difference in the percentages of intra- and extracellular NA' trypomastigotes could be due to the loss of the epitope recognized by the TCN-2 following parasite exiting from the host cell, we compared neuraminidases obtained from intra- and extracellular parasites. Immunoprecipitation of metabolically labeled enzyme with TCN-2 demonstrated that neuraminidase from intracellular (Fig. 2A) and extracellular (Fig. 2B) trypomastigotes displayed the same molecular polymorphism characteristic of the Silvio X-10/4 strain (7). In addition, the NA' subset of parasites was first described by using a polyclonal anti-neuraminidase antibody (1) which should recognize many epitopes on the neuraminidase molecule. Precipitates of amastigote lysates (Fig. 2C) and of trypomastigote lysates with an isotype-matched control monoclonal antibody (Fig. 2D) were negative for neuraminidase, confirming that the enzyme is developmentally regulated and that the binding of TCN-2 is specific.
In view of these results we propose the following differentiation pathway for T. cruzi cultivated in tissue culture: amastigote -- intracellular NA' trypomastigotes -> extracellular NA- and NA+ trypomastigotes. The results presented here confirm that neuraminidase is expressed on transformation of amastigotes into trypomastigotes. All newly transformed trypomastigotes express the enzyme. Since the majority of the trypomastigotes become NA- in the extracellular environment, we hypothesize that T. cruzi neuraminidase may play a role in the exiting of the trypomastigotes from the infected cells. This hypothesis is consistent with one of the possible functions of the influenza virus neuraminidase, which is to keep viral particles in a desialylated, disaggregated state and consequently to facilitate viral budding from infected cells (2). Whether T. cruzi neuraminidase aids trypanosoma exiting from infected cells can only be ascertained by future experiments. However, it is clear that the neuraminidase is a useful marker to study the intracellular differentiation of amastigote into trypomastigote and the extracellular maturation' of trypomastigotes. The use of a specific probe in this experimentally accessible developmental system holds exciting prospects for learning more about the molecular and cellular processes controlling differentiation and transformation of T. cruzi. This work was supported by NIH grant Al 18102 (to M.E.A.P.) and NIH postdoctoral fellowship Al 07380 (to I.R.). REFERENCES 1. Cavallesco, C., and M. E. A. Pereira. 1988. Antibody to Trypanosoma cruzi neuraminidase enhances infection in vitro and identifies a subpopulation of trypomastigotes. J. Immunol. 140:617625. 2. Colman, P. M., and C. W. Ward. 1985. Structure and diversity of Influenza virus neuraminidase. Curr. Top. Microbiol. Immunol. 114:177-255. 3. Libby, P., J. Alroy, and M. E. A. Pereira. 1986. A neuraminidase from Trypanosoma cruzi removes sialic acid from the surface of mammalian myocardial and endothelial cells. J. Clin. Invest. 77:127-135. 4. Pereira, M. E. A. 1983. A developmentally regulated neuraminidase activity in Trypanosoma cruzi. Science 219:1444-1446. 5. Pereira, M. E. A., and R. Hoff. 1986. Heterogeneous distribution of neuraminidase activity in strains and clones of Trypanosoma cruzi and its possible association with parasite myotropism. Mol. Biochem. Parasitol. 20:183-189. 6. Postan, M., J. A. Dvorak, and J. P. McDaniel. 1983. Studies on Trypanosoma cruzi clones in inbred mice. I. A comparison of the course of infection of C3H/HEN mice with two clones isolated from a common source. Am. J. Trop. Med. Hyg. 32:497-506. 7. Prioli, R. P., J. S. Mejia, and M. E. A. Pereira. 1990. Monoclonal antibodies against Trypanosoma cruzi neuraminidase reveal enzyme polymorphism, recognize a subset of trypomastigotes and enhance infection in vitro. J. Immunol. 144:4384-4391. 8. Siekevitz, M., M. L. Gefter, P. Brodeur, and A. MarshakRothstein. 1982. The genetic basis of antibody production: the dominant anti-arsonate idiotype response of the strain A mouse. Eur. J. Immunol. 12:1023-1032. 9. Titto, E. H., and F. G. Araujo. 1988. Serum neuraminidase activity and hematological alterations in acute human Chagas' disease. Clin. Immunol. Immunopathol. 46:157-161.
