EXPERIMENTAL
PARASITOLOGY
71, 241-244 (1990)
MINIREVIEW The Acid-Active Hemolysin of Trypanosoma cruzi NORMA W. ANDREWS Department
of Pathology,
ANDREWS,
Parasitology
New York Universio
Medical
Center, New York, New York 10016, U.S.A.
N. W. 1990. The acid-active hemolysin of Trypanosoma
cruzi. Experimental
71, 241-244. 8 1990 Academic press. Inc.
INTRODUCTION
Intracellular parasites enter host cells, in most cases, within vacuolar compartments. Enveloped animal viruses, that depend on host cell machinery for replication, deliver their genome to the cytosol by membrane fusion, mediated by conformational changes that their spike glycoproteins undergo at the low pH of endosomes (Marsh and Helenius 1989). Protozoans, as eukaryotic cells, can survive in relative independence from the host cell and have developed different intracellular survival strategies. Leishmaniu, for example, multiplies inside functional phagolysosomes of macrophages (Antoine et al. 1987) and somehow evades the killing mechanisms that are normally effective in these cells. Toxoplasma gonu’ii invades cells and replicates within modified vacuoles that do not acidify and to which lysosomes do not fuse (Sibley et al. 1985). Trypunosomu cruzi (Ley et al. 1990), like Theileriu (Fawcett ef al. 1982) and Bubesiu (Rudzinska 1981), leaves the phagolysosome soon after cell invasion and multiplies free in the cytosol. A common feature of these and many other parasites is that all must disrupt both vacuolar and host cell plasma membranes at some point in their intracellular cycle. This review will focus on recent observations made with T. cruzi which led to the identification of a secreted membranolytic protein active at low pH. Evidence suggesting that this protein mediates phagosome disruption (and perhaps also host cell lysis)
by forming transmembrane pores will be discussed. OBSERVATION OF HEMOLYTIC AND FUSOGENIC ACTIVITY ASSOCIATED WITH T. cruzi EPIMASTIGOTES
Previous reports of hemolytic activity associated with T. cruzi were made by Calderon et al. (1986, 1989). These investigators observed that chicken and human erythrocytes were lysed when incubated with epimastigotes (the extracellular, insect stages of T. cruzi) in the exponential phase of growth. Parasites from stationary phase cultures were reported to induce erythrocyte fusion before lysis, a property presumably associated with the presence of infective metacyclic trypomastigotes in the cultures. Trypsin digestion of parasite surface proteins abolished the fusogenic ability but not hemolysis, an observation that led the authors to suggest that the hemolytic agent was probably deeply embedded on the parasite’s plasma membrane. A HEMOLYTIC PROTEIN OPTIMALLY ACTIVE AT pH 5.5 Is SECRETED BY T. cruzi
A heat-labile, trypsin-sensitive molecule that lysed erythocytes from several animal species was later found in T. cruzi supematants (Andrews and Whitlow 1989). Parasite-target cell contact was not required for lysis, and production of the hemolysin was blocked by metabolic inhibitors, indicating that it was a secretory product. The activity
241 0014-4894/90$3.00 Copyright 0 1990 by Academic Ress, Inc. Ail rights of reproduction in any form reserved.
242
MINIREVIEW:
ACID-ACTIVE
was optimal at pH 5.5, which immediately suggested that the hemolysin could have a role in intracellular acidic compartments. Comparison of the three developmental stages indicated that amastigotes, the intracellular forms, were the most hemolytically active, followed by trypomastigotes, the infective bloodstream forms. Some activity, that required longer incubation periods for detection, was observed with epimastigotes. No hemolysis was induced by all parasite stages at neutral pH, and neutralization of parasite supematants irreversibly inactivated the hemolysin. These observations raised questions regarding the identity between this acid pH-dependent secreted hemolysin and the hemolytic factor mentioned above, described by Calderon et al. (1986). Although those investigators performed their assays at pH 7.4, it is conceivable that the medium became acidified during the course of their experiments, as it is known to occur as a consequence of parasite metabolic activity. THE ESCAPE OF PHAGOLYSOSOME RAISING ACIDIC
T. cruzi FROM Is INHIBITED THE pH
THE BY
OF
COMPARTMENTS
Several studies had demonstrated that lysosomes fused with trypomastigotecontaining vacuoles soon after host cell invasion and that 24 hr later, amastigotes were free in the cytosol (Kress et al. 1975; Nogueira and Cohn 1976; Milder and Kloetzel 1980; Ulisses de Carvalho and de Souza 1989). However, until recently it was not clear how long after cell entry and at which developmental stage the parasites escaped from the phagosome. Ley et al. (1988), confirming and expanding previous reports by McCabe et al. (1984) and Carvalho and de Souza (1986), showed that amastigotes could enter mammalian cells, escape from the phagosome, and multiply in the cytosol, thus sustaining a complete infective cycle. This observation raised the question of whether trypomastigotes first had to trans-
HEMOLYSIN
form into amastigotes before inducing phagosome disruption. Earlier studies by Dvorak and Hyde (1973) estimated the period cf transformation of trypomastigotes into anastigotes following host cell invasion as a,?proximately 3 hr. These studies, however, ilid not determine if the change occurred in or out of the phagosome. Recent electron microscopic observations by Ulisses de Carvalho and de Souza (1989) and Ley et al. (1990) clarified the issue, demonstrating that trypomastigotes can leave the phagosome before complete transformation into amastigotes and that this occurs within the first 2 hr after host cell invasion. Ley et al. (1990) also showed that the tight vacuoles in which the parasites reside intracellularly at early stages are acidic compartments that can be labeled by the acidotropic compound DAMP (Anderson et al. 1984). Agents that raise the pH of acidic compartments such as ammonium chloride, chloroquine, methylamine, or monensin markedly decreased the number of T. cruzi escaping from the phagosomes into the cytosol (Ley et al. 1990). These observations provided supportive evidence for the hypothesis (Andrews and Whitlow 1989) postulating that the acid-active hemolysin secreted by T. cruzi was the agent responsible for disruption of the phagosome membrane. THE
HEMOLYSIN
CHANNELS
FORMS
ON TARGET
LARGE
MEMBRANES
Sieving experiments with erythrocyte ghosts indicated that the hemolysin formed large lesions on target membranes that allowed the passage of dextran molecules of an estimated molecular diameter of 10 nm (Andrews and Whitlow 1989). Since only a population of smaller dextran molecules was released while the larger ones were retained inside the ghosts, it was concluded that lysis was most likely due to the formation of transmembrane channels and not to membrane disintegration by a phospholipase or detergent-like molecule. Similar
ACID-ACTIVE
sieving effects are generally observed with bacterial pore-forming toxins and also with the membrane attack complex formed by the terminal components of complement. Recent studies demonstrated that acidified T. cruzi supernatants are also cytotoxic for nucleated cells (Andrews et al. 1990). This and the earlier observation that the channels formed on target membranes were large suggested a possible analogy between the mechanism of action of the T. cruzi hemolysin and that of the membrane attack complex of complement. The last component of the membrane attack complex, C9, shares several properties with perforin, the pore-forming protein of cytotoxic lymphocytes. These include immunological crossreactivity, molecular mass (both migrate as 60-66 kDa nonreduced and 70-75 kDa reduced), polymerization into membranespanning tubules of a large internal diameter, and cytotoxic activity for nucleated cells (Young and Cohn 1987). Recent studies indicate that the T. cruzi hemolysin copurifies with a protein of 60-75 kDa immunologically related to human C9 and that fractions enriched in this protein form ion channels in planar phospholipid bilayers at low pH (Andrews et al. 1990). Work in progress aims at verifying if the T. cruzi hemolysin has structural similarities with this class of eukaryotic pore-forming proteins that includes C9 and perforin. CONCLUDING
REMARKS
243
HEMOLYSIN
system in human chagasic patients and postulated that the pathology of the chronic phase was a consequence of defective tissue innervation. A putative neurotoxin produced by the parasites was proposed as the agent responsible for neuronal destruction. It is tempting to speculate that the acidactive hemolysin discussed here may correspond to the toxin proposed by Koberle. An understanding of its mechanism of action may clarify issues, so far obscure, related to the pathogenesis of Chagas’ disease. REFERENCES ANDERSON, R. G. W., FALK, J. R., GOLDSTEIN, J. L., AND BROWN, M. S. 1984. Visualization of acidic organelles in intact cells by electron microscopy. Proceedings of the National Academy of Sciences USA 81: 4838-4842.
ANDREWS, N. W., AND WHITLOW, M. B. 1989. Secretion by Trypanosoma cruzi of a hemolysin active at low pH. Molecular and Biochemical Parasitology 33, 249-256. ANDREWS, N. W., ABRAMS, C. N., SLATIN, S. L., AND GRIFFITHS, G. 1990. A T. cruzi-secreted protein immunologically related to the complement component C9: Evidence for membrane poreforming activity at low pH. Cell, in press. ANTOINE, J. C., JOUANNE, C., RYTER, A., AND ZILBERFAREI,V. 1987. Leishmania mexicana: A cytochemical and quantitative study of lysosomal enzymes in infected rat bone marrow macrophages. Experimental
Parasitology
64, 485-498.
CALDERON, R. O., AGUERRI, A. M., AND BRONIA, D. H. 1986. Trypanosoma cruzi: Variable fusogenic ability by different growth phases of the epimastigote form. Experimental Parasitology 62, 453-455. CALDERON, R. O., LUJAN, H. D., AGUERRI, A. M., AND BRONIA, D. H. 1989. Trypanosoma cruzi: Involvement of proteolytic activity during cell fusion induced by epimastigote form. Molecular and Cellular Biochemistry 86, 189-200. CARVALHO,T. U., AND DE SOUZA,W. 1986. Infectivity of amastigotes of Trypanosoma cruzi. Revista do
T. cruzi is the causative agent of Chagas’ disease, a serious condition that affects millions of people in Central and South America. A proportion of the infected individuals develop cardiomyopathy, megacolon, and megaesophagus during the chronic stage of Instituto de Medicina Tropical de Sao Paulo 28, the disease, many years after the initial in205-210. fection. The cause of these abnormalities is DVORAK,J. A., AND HYDE, T. P. 1973. Trypanosoma cruzi: Interaction with vertebrate cells in vitro. 1. poorly understood since they occur at a Individual interactions at the cellular and subcellular stage in which there is no intense intracellevels. Experimental Parasitology 34, 268-283. lular parasite proliferation. Koberle (1968) FAWCETT, D. W., DOXSEY, S., STAGG, D. A., AND described significant reductions in the numYOUNG, A. S. 1982. The entry of sporozoites of Theileria parva into bovine lymphocytes in vitro: ber of neurons of the autonomic nervous
244
MINIREVIEW:
ACID-ACTIVE
Electron microscopic observations. European Jour27, 10-21. KOBERLE, F. 1968. Chagas’ disease and Chagas’ syndrome: The pathology of American trypanosomiasis. Advances in Parasitology 6, 63-l 16. KRESS,Y., BLOOM,B. R., WIDENER,M., ROWEN,A., AND TANOWITZ, H. 1975. Resistance of Trypanosoma cruzi to killing by macrophages. Nature (Lonnal of Cell Biology
don) 257, 3943%.
LEY, V., ANDREWS, N. W., ROBBINS, E. S., AND NUSSENZWEIG,V. 1988. Amastigotes of Trypanosoma cruzi sustain an infective cycle in mammalian cells. Journal of Experimental Medicine 168, 649659. LEY, V., ROBBINS, E. S., NUSSENZWEIG,V., AND ANDREWS, N. W. 1990. The exit of Trypanosoma cruzi from the phagosome is inhibited by raising the pH of acidic compartments. Journal of Experimental Medicine 171, 401-413. MARSH, M., AND HELENIUS, A. 1989.Virus entry into animal cells. Advances in Virus Research 36, 107151. MCCABE, R. E., REMINGTON, J. S., AND ARAUJO,
F. G. 1984. Mechanisms of invasion and replication of the intracellular stage in Trypanosoma cruzi. Infection
and Immunity
46, 372-376.
HEMOLYSIN
MILDER, R., AND KLOETZEL, J. 1980. The develop ment of Trypanosoma cruzi in macrophages in vitro: Interaction with lysosomes and host cell fate. Parasitology 80, 139-145. NOGUEIRA, N., AND COHN, Z. A. 1976. Trypanosoma cruzi: Mechanism of entry and intracellular fate in mammalian cells. Journal of Experimental Medicine 143, 1402-1420.
RUDZINSKA, M. A. 1981. Morphological aspects of host cell-parasite relationships in babesiosis. In “Babesiosis” (M. Ristic and J. P. Kreier, Eds.), pp. 87-141. Academic Press, New York. SIBLEY, L. D., WEINER, E., AND KRAHENBUHL, J. L.
1985. Phagosome acidification blocked by intracellular Toxoplasma gondii. Nature (London) 351,416 419.
ULISSESDE CARVALHO, T. M., AND DE SOUZA, W. 1989. Early events related with the behavior of Trypanosoma cruzi within an endocytic vacuole in mouse peritoneal macrophages. Cell Structure and Function 14, 383-392. YOUNG, J. D.-E., AND COHN, Z. A. 1987. Cellular and
humoral mechanisms of cytotoxicity: Structural and functional analogies. Advances in Immunology 41, 269-332. Received 13 April 1990; accepted 17 April 1990
PARASITOLOGY
71, 241-244 (1990)
MINIREVIEW The Acid-Active Hemolysin of Trypanosoma cruzi NORMA W. ANDREWS Department
of Pathology,
ANDREWS,
Parasitology
New York Universio
Medical
Center, New York, New York 10016, U.S.A.
N. W. 1990. The acid-active hemolysin of Trypanosoma
cruzi. Experimental
71, 241-244. 8 1990 Academic press. Inc.
INTRODUCTION
Intracellular parasites enter host cells, in most cases, within vacuolar compartments. Enveloped animal viruses, that depend on host cell machinery for replication, deliver their genome to the cytosol by membrane fusion, mediated by conformational changes that their spike glycoproteins undergo at the low pH of endosomes (Marsh and Helenius 1989). Protozoans, as eukaryotic cells, can survive in relative independence from the host cell and have developed different intracellular survival strategies. Leishmaniu, for example, multiplies inside functional phagolysosomes of macrophages (Antoine et al. 1987) and somehow evades the killing mechanisms that are normally effective in these cells. Toxoplasma gonu’ii invades cells and replicates within modified vacuoles that do not acidify and to which lysosomes do not fuse (Sibley et al. 1985). Trypunosomu cruzi (Ley et al. 1990), like Theileriu (Fawcett ef al. 1982) and Bubesiu (Rudzinska 1981), leaves the phagolysosome soon after cell invasion and multiplies free in the cytosol. A common feature of these and many other parasites is that all must disrupt both vacuolar and host cell plasma membranes at some point in their intracellular cycle. This review will focus on recent observations made with T. cruzi which led to the identification of a secreted membranolytic protein active at low pH. Evidence suggesting that this protein mediates phagosome disruption (and perhaps also host cell lysis)
by forming transmembrane pores will be discussed. OBSERVATION OF HEMOLYTIC AND FUSOGENIC ACTIVITY ASSOCIATED WITH T. cruzi EPIMASTIGOTES
Previous reports of hemolytic activity associated with T. cruzi were made by Calderon et al. (1986, 1989). These investigators observed that chicken and human erythrocytes were lysed when incubated with epimastigotes (the extracellular, insect stages of T. cruzi) in the exponential phase of growth. Parasites from stationary phase cultures were reported to induce erythrocyte fusion before lysis, a property presumably associated with the presence of infective metacyclic trypomastigotes in the cultures. Trypsin digestion of parasite surface proteins abolished the fusogenic ability but not hemolysis, an observation that led the authors to suggest that the hemolytic agent was probably deeply embedded on the parasite’s plasma membrane. A HEMOLYTIC PROTEIN OPTIMALLY ACTIVE AT pH 5.5 Is SECRETED BY T. cruzi
A heat-labile, trypsin-sensitive molecule that lysed erythocytes from several animal species was later found in T. cruzi supematants (Andrews and Whitlow 1989). Parasite-target cell contact was not required for lysis, and production of the hemolysin was blocked by metabolic inhibitors, indicating that it was a secretory product. The activity
241 0014-4894/90$3.00 Copyright 0 1990 by Academic Ress, Inc. Ail rights of reproduction in any form reserved.
242
MINIREVIEW:
ACID-ACTIVE
was optimal at pH 5.5, which immediately suggested that the hemolysin could have a role in intracellular acidic compartments. Comparison of the three developmental stages indicated that amastigotes, the intracellular forms, were the most hemolytically active, followed by trypomastigotes, the infective bloodstream forms. Some activity, that required longer incubation periods for detection, was observed with epimastigotes. No hemolysis was induced by all parasite stages at neutral pH, and neutralization of parasite supematants irreversibly inactivated the hemolysin. These observations raised questions regarding the identity between this acid pH-dependent secreted hemolysin and the hemolytic factor mentioned above, described by Calderon et al. (1986). Although those investigators performed their assays at pH 7.4, it is conceivable that the medium became acidified during the course of their experiments, as it is known to occur as a consequence of parasite metabolic activity. THE ESCAPE OF PHAGOLYSOSOME RAISING ACIDIC
T. cruzi FROM Is INHIBITED THE pH
THE BY
OF
COMPARTMENTS
Several studies had demonstrated that lysosomes fused with trypomastigotecontaining vacuoles soon after host cell invasion and that 24 hr later, amastigotes were free in the cytosol (Kress et al. 1975; Nogueira and Cohn 1976; Milder and Kloetzel 1980; Ulisses de Carvalho and de Souza 1989). However, until recently it was not clear how long after cell entry and at which developmental stage the parasites escaped from the phagosome. Ley et al. (1988), confirming and expanding previous reports by McCabe et al. (1984) and Carvalho and de Souza (1986), showed that amastigotes could enter mammalian cells, escape from the phagosome, and multiply in the cytosol, thus sustaining a complete infective cycle. This observation raised the question of whether trypomastigotes first had to trans-
HEMOLYSIN
form into amastigotes before inducing phagosome disruption. Earlier studies by Dvorak and Hyde (1973) estimated the period cf transformation of trypomastigotes into anastigotes following host cell invasion as a,?proximately 3 hr. These studies, however, ilid not determine if the change occurred in or out of the phagosome. Recent electron microscopic observations by Ulisses de Carvalho and de Souza (1989) and Ley et al. (1990) clarified the issue, demonstrating that trypomastigotes can leave the phagosome before complete transformation into amastigotes and that this occurs within the first 2 hr after host cell invasion. Ley et al. (1990) also showed that the tight vacuoles in which the parasites reside intracellularly at early stages are acidic compartments that can be labeled by the acidotropic compound DAMP (Anderson et al. 1984). Agents that raise the pH of acidic compartments such as ammonium chloride, chloroquine, methylamine, or monensin markedly decreased the number of T. cruzi escaping from the phagosomes into the cytosol (Ley et al. 1990). These observations provided supportive evidence for the hypothesis (Andrews and Whitlow 1989) postulating that the acid-active hemolysin secreted by T. cruzi was the agent responsible for disruption of the phagosome membrane. THE
HEMOLYSIN
CHANNELS
FORMS
ON TARGET
LARGE
MEMBRANES
Sieving experiments with erythrocyte ghosts indicated that the hemolysin formed large lesions on target membranes that allowed the passage of dextran molecules of an estimated molecular diameter of 10 nm (Andrews and Whitlow 1989). Since only a population of smaller dextran molecules was released while the larger ones were retained inside the ghosts, it was concluded that lysis was most likely due to the formation of transmembrane channels and not to membrane disintegration by a phospholipase or detergent-like molecule. Similar
ACID-ACTIVE
sieving effects are generally observed with bacterial pore-forming toxins and also with the membrane attack complex formed by the terminal components of complement. Recent studies demonstrated that acidified T. cruzi supernatants are also cytotoxic for nucleated cells (Andrews et al. 1990). This and the earlier observation that the channels formed on target membranes were large suggested a possible analogy between the mechanism of action of the T. cruzi hemolysin and that of the membrane attack complex of complement. The last component of the membrane attack complex, C9, shares several properties with perforin, the pore-forming protein of cytotoxic lymphocytes. These include immunological crossreactivity, molecular mass (both migrate as 60-66 kDa nonreduced and 70-75 kDa reduced), polymerization into membranespanning tubules of a large internal diameter, and cytotoxic activity for nucleated cells (Young and Cohn 1987). Recent studies indicate that the T. cruzi hemolysin copurifies with a protein of 60-75 kDa immunologically related to human C9 and that fractions enriched in this protein form ion channels in planar phospholipid bilayers at low pH (Andrews et al. 1990). Work in progress aims at verifying if the T. cruzi hemolysin has structural similarities with this class of eukaryotic pore-forming proteins that includes C9 and perforin. CONCLUDING
REMARKS
243
HEMOLYSIN
system in human chagasic patients and postulated that the pathology of the chronic phase was a consequence of defective tissue innervation. A putative neurotoxin produced by the parasites was proposed as the agent responsible for neuronal destruction. It is tempting to speculate that the acidactive hemolysin discussed here may correspond to the toxin proposed by Koberle. An understanding of its mechanism of action may clarify issues, so far obscure, related to the pathogenesis of Chagas’ disease. REFERENCES ANDERSON, R. G. W., FALK, J. R., GOLDSTEIN, J. L., AND BROWN, M. S. 1984. Visualization of acidic organelles in intact cells by electron microscopy. Proceedings of the National Academy of Sciences USA 81: 4838-4842.
ANDREWS, N. W., AND WHITLOW, M. B. 1989. Secretion by Trypanosoma cruzi of a hemolysin active at low pH. Molecular and Biochemical Parasitology 33, 249-256. ANDREWS, N. W., ABRAMS, C. N., SLATIN, S. L., AND GRIFFITHS, G. 1990. A T. cruzi-secreted protein immunologically related to the complement component C9: Evidence for membrane poreforming activity at low pH. Cell, in press. ANTOINE, J. C., JOUANNE, C., RYTER, A., AND ZILBERFAREI,V. 1987. Leishmania mexicana: A cytochemical and quantitative study of lysosomal enzymes in infected rat bone marrow macrophages. Experimental
Parasitology
64, 485-498.
CALDERON, R. O., AGUERRI, A. M., AND BRONIA, D. H. 1986. Trypanosoma cruzi: Variable fusogenic ability by different growth phases of the epimastigote form. Experimental Parasitology 62, 453-455. CALDERON, R. O., LUJAN, H. D., AGUERRI, A. M., AND BRONIA, D. H. 1989. Trypanosoma cruzi: Involvement of proteolytic activity during cell fusion induced by epimastigote form. Molecular and Cellular Biochemistry 86, 189-200. CARVALHO,T. U., AND DE SOUZA,W. 1986. Infectivity of amastigotes of Trypanosoma cruzi. Revista do
T. cruzi is the causative agent of Chagas’ disease, a serious condition that affects millions of people in Central and South America. A proportion of the infected individuals develop cardiomyopathy, megacolon, and megaesophagus during the chronic stage of Instituto de Medicina Tropical de Sao Paulo 28, the disease, many years after the initial in205-210. fection. The cause of these abnormalities is DVORAK,J. A., AND HYDE, T. P. 1973. Trypanosoma cruzi: Interaction with vertebrate cells in vitro. 1. poorly understood since they occur at a Individual interactions at the cellular and subcellular stage in which there is no intense intracellevels. Experimental Parasitology 34, 268-283. lular parasite proliferation. Koberle (1968) FAWCETT, D. W., DOXSEY, S., STAGG, D. A., AND described significant reductions in the numYOUNG, A. S. 1982. The entry of sporozoites of Theileria parva into bovine lymphocytes in vitro: ber of neurons of the autonomic nervous
244
MINIREVIEW:
ACID-ACTIVE
Electron microscopic observations. European Jour27, 10-21. KOBERLE, F. 1968. Chagas’ disease and Chagas’ syndrome: The pathology of American trypanosomiasis. Advances in Parasitology 6, 63-l 16. KRESS,Y., BLOOM,B. R., WIDENER,M., ROWEN,A., AND TANOWITZ, H. 1975. Resistance of Trypanosoma cruzi to killing by macrophages. Nature (Lonnal of Cell Biology
don) 257, 3943%.
LEY, V., ANDREWS, N. W., ROBBINS, E. S., AND NUSSENZWEIG,V. 1988. Amastigotes of Trypanosoma cruzi sustain an infective cycle in mammalian cells. Journal of Experimental Medicine 168, 649659. LEY, V., ROBBINS, E. S., NUSSENZWEIG,V., AND ANDREWS, N. W. 1990. The exit of Trypanosoma cruzi from the phagosome is inhibited by raising the pH of acidic compartments. Journal of Experimental Medicine 171, 401-413. MARSH, M., AND HELENIUS, A. 1989.Virus entry into animal cells. Advances in Virus Research 36, 107151. MCCABE, R. E., REMINGTON, J. S., AND ARAUJO,
F. G. 1984. Mechanisms of invasion and replication of the intracellular stage in Trypanosoma cruzi. Infection
and Immunity
46, 372-376.
HEMOLYSIN
MILDER, R., AND KLOETZEL, J. 1980. The develop ment of Trypanosoma cruzi in macrophages in vitro: Interaction with lysosomes and host cell fate. Parasitology 80, 139-145. NOGUEIRA, N., AND COHN, Z. A. 1976. Trypanosoma cruzi: Mechanism of entry and intracellular fate in mammalian cells. Journal of Experimental Medicine 143, 1402-1420.
RUDZINSKA, M. A. 1981. Morphological aspects of host cell-parasite relationships in babesiosis. In “Babesiosis” (M. Ristic and J. P. Kreier, Eds.), pp. 87-141. Academic Press, New York. SIBLEY, L. D., WEINER, E., AND KRAHENBUHL, J. L.
1985. Phagosome acidification blocked by intracellular Toxoplasma gondii. Nature (London) 351,416 419.
ULISSESDE CARVALHO, T. M., AND DE SOUZA, W. 1989. Early events related with the behavior of Trypanosoma cruzi within an endocytic vacuole in mouse peritoneal macrophages. Cell Structure and Function 14, 383-392. YOUNG, J. D.-E., AND COHN, Z. A. 1987. Cellular and
humoral mechanisms of cytotoxicity: Structural and functional analogies. Advances in Immunology 41, 269-332. Received 13 April 1990; accepted 17 April 1990
