Role of cell surface mannose residues in host cell invasion by Trypanosoma cruzi.

The role of mannose residues on the membranes of Trypanosoma cruzi and its host cells in their association (surface binding plus internalization of the parasite) leading to infection was studied. Used in this work were the bloodstream (trypomastigote), intracellular (amastigote) and insect-transmissible (metacyclic trypomastigote) forms of the parasite; mouse macrophages and rat heart myoblasts were used as the host cells. Removal of mannose residues from the surface of all forms of the parasite by treatment with alpha-mannosidase produced a marked increase in their respective abilities to associate with either host cell. The increase was more pronounced with the bloodstream and insect-derived trypomastigotes (which can penetrate cell membranes) than with the amastigotes (which can not do so). By contrast, mannosidase treatment of the macrophages and the myoblasts caused a significant decrease in the ability of these cells to associate with either bloodstream or insect-derived trypomastigote forms. The capacity of mannosidase-treated macrophages to take up the non-invasive amastigotes was also reduced. These results, as a whole, suggest that mannose residues on the surface of the parasite modulate their binding to macrophages and myoblasts and that mannose residues on the surface of these host cells play a role in cell association with the parasite.

Role of surface N-acetylglucosamine residues on host cell infection by Trypanosoma cruzi.

We studied the role of surface GlcNAc residues on the surface of invasive (mouse-blood and insect-derived trypomastigotes) and non-invasive amastigote forms of Trypanosoma cruzi on parasite association with (i.e., surface binding plus internalization) macrophages and heart myoblasts. Removal of GlcNAc from the three forms of the parasite with beta-N-acetylglucosaminidase markedly increased the number of organisms per 100 cells and caused the organisms to associate with a greater percentage of host cells. N-Acetylglucosaminidase did not produce this effect after heat-inactivation and a substrate of the enzyme, N,N'-diacetylchitobiose, reduced it when it was present during the enzymatic treatment. The N-acetylglucosaminidase effect on T. cruzi was reversible after 2.5 h. When macrophages or myoblasts were treated with N-acetylglucosaminidase, their capacities to associate with blood or insect-derived trypomastigotes was reduced. Since removal of GlcNAc residues from the parasite surface increased their association with the host cells, GlcNAc would appear to interfere with the association process. On the other hand, GlcNAc residues on the host cell appear to favor the association.

The effect of swainsonine on the association of Trypanosoma cruzi with host cells.

The role of glycoprotein processing in Trypanosoma cruzi association with mammalian host cells (i.e., surface binding and internalization) was studied by using swainsonine - an inhibitor of glycoprotein processing. The presence of swainsonine in co-cultures of blood forms of T. cruzi with either mouse peritoneal macrophages or rat heart myoblasts markedly reduced parasite-host cell association as evidenced by significant decreases in both the percentage of cells associating with the organisms and the number of parasites per cell. This inhibition appeared to result from effects on both the parasite and the host cells since pretreatment of either one reduced its association with the untreated counterpart. The inhibitory effect of swainsonine on the host cells was demonstrable with trypomastigote forms derived from either infected mouse blood or the feces of infected insect vectors, and also with amastigotes. Conversely, treatment of the parasites with swainsonine inhibited their association with the host cells. Studies with blood trypomastigotes revealed that the swainsonine effect was reversible within 3 h. Two observations suggested that swainsonine had induced the parasite to produce defective oligosaccharides rich in mannose. First, swainsonine-treated organisms displayed a greater capacity to bind concanavalin than medium-treated organisms. Second, presence of alpha-methyl mannoside reduced such increased concanavalin A binding. Taken together, the present results suggest important roles for glycoprotein processing and oligosaccharide structures on the surface of both T. cruzi and its host cells in the initial stages of host cell infection.

N,N'-thiophene-substituted polyamine analogs inhibit mammalian host cell invasion and intracellular multiplication of Trypanosoma cruzi.

We studied the effects of two N,N'-thiophene-substituted polyamine analogs (MDL 28302 and MDL 29431) on the capacities of Trypanosoma cruzi, the etiologic agent of Chagas' disease, to invade and multiply within a mammalian host cell. Both compounds inhibited infectivity significantly in a time- and concentration-dependent manner. This inhibition resulted from a selective effect on the parasite, because pretreatment of T. cruzi but not host cell cultures with either MDL 28302 or MDL 29431 reduced infectivity. The parasite gradually recovered its infective capacity after removal of unincorporated polyamine analog, denoting the reversible nature of the inhibitory effect. Some biochemical modification of MDL 28302 and MDL 29431 appeared to be required for their inhibitory activities to be exerted, since the effects of these drugs on T. cruzi infectivity were abrogated by MDL 72527, a drug known to inhibit polyamine oxidase (PAO) activity specifically. Supporting the notion of that products of MDL 28302 and MDL 29431 oxidation by PAO were involved in the activity of these compounds was the finding that PAO competitive substrates (N1-acetylspermine and N1-acetylspermidine) also abolished the inhibition of T. cruzi infectivity mediated by MDL 28302 or MDL 29431. However, we can not rule out that MDL 72527 and the PAO competitive substrates might have altered an alternative mechanism because no significant polyamine oxidase activity could be demonstrated in preparations of lysed or intact T. cruzi in assays monitoring conversion of [14C]spermine to [14C]spermidine. When either MDL 28302 or MDL 29431 was added to infected cell cultures, a marked reduction in the rate of intracellular parasite growth ensued.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of p-chloromercuriphenylsulfonic acid on Trypanosoma cruzi infection of mammalian host cells in vitro.

Treatment of Trypanosoma cruzi blood trypomastigotes with p-chloromercuriphenylsulfonic acid (PCMS) increased the association of the parasite with either mouse resident peritoneal macrophages or rat heart myoblasts in vitro. The effect was evidenced by elevation of both the percentage of host cells with parasites and the number of flagellates per 100 cells. The effect of PCMS appeared to be largely on the process of parasite penetration rather than surface binding as it was seen at 37 degrees C but not at 4 degrees C. A short pretreatment time, 5 min, was sufficient to elicit the enhancement, suggesting that the primary effect of PCMS might be at the parasite's cell surface. The PCMS effect was reversible as the parasite returned to normal levels of association with the host cells in less than 4 h after removal of excess PCMS. That sulfhydryl groups were involved in the PCMS effect was indicated by the abilities of excess cysteine and glutathione to block it. These results suggest a role for free sulfhydryl groups on the parasite surface in the process of host cell invasion.

Role of membrane N-linked oligosaccharides in host cell interaction with invasive forms of Trypanosoma cruzi.

The removal of N-linked oligosaccharides by peptide-N4-[N-acetyl-beta-glucoseaminyl]asparagine amidase (previously known as aspartoglycosylamine amidohydrolase and abbreviated N-glycanase) from the surface of blood or insect-transmissible forms of Trypanosoma cruzi markedly increased the capacity of these organisms to associate with (i.e., bind and penetrate) either mouse peritoneal macrophages or rat heart myoblasts. This effect was evidenced by a significant elevation in both the percentage of infected host cells and the average number of parasites per 100 cells. Conversely, N-glycanase treatment of either host cell markedly reduced both parameters to levels significantly below those obtained with cells mock treated with medium alone. The N-glycanase effect on the parasites was inhibited by heat inactivation of the enzyme or by the presence of fetuin, an N-glycanase substrate. The enhanced capacity of N-glycanase-treated T. cruzi to engage the host cells started to subside 2 h after the treatment, indicating the reversibility of the effect. The decreased reactivity of N-glycanase-treated macrophages or myoblasts with T. cruzi suggests that N-linked oligosaccharides on these host cells are involved in the initial phase of the cell infection process. Instead, because T. cruzi interacted more effectively with host cells after treatment with N-glycanase, parasite surface N-linked oligosaccharides would seem to interfere with the association.

Effects of leukotriene C4 on macrophage association with and intracellular fate of Trypanosoma cruzi.

Leukotriene C4 (LTC4) enhanced the association of mouse peritoneal macrophages (MPM) with Trypanosoma cruzi, increasing the proportion of MPM associating with parasites and the number of trypanosomes per MPM. LTC4 affected both cells since pretreatment of either one increased the association. LTC4 also enhanced MPM uptake of killed T. cruzi or latex beads, denoting stimulation of phagocytosis. However, since LTC4 pretreatment of rat heart myoblasts--nonphagocytic cells--also increased the association, host cell membrane alterations induced by LTC4 may also facilitate parasite invasion. Inhibition of MPM guanylate cyclase abrogated the LTC4 effect, suggesting a role for elevated levels of cyclic GMP. LTC4 also increased the rate of intracellular parasite killing by MPM. These results suggest that LTC4, occurring in inflammation such as develops in T. cruzi infection, regulates parasite clearance by MPM by increasing uptake and intracellular destruction.

Increased host cell-Trypanosoma cruzi interaction following phospholipase D treatment of the parasite surface.

We examined the effect of phospholipase D (PLD) treatment on the ability of Trypanosoma cruzi to interact with phagocytic and nonphagocytic host cells. The presence of PLD during the incubation of parasites with mouse peritoneal macrophages caused significant increases in both the number of parasites per 100 macrophages and the percentage of macrophages associated with parasites. Parasites pretreated with PLD, washed, and then incubated with untreated macrophages showed a marked increase in parasite-host cell association. In contrast, when only the macrophages were pretreated with PLD, there was no significant change in the association. Parasites required 45 min of PLD treatment before a significant enhancement in parasite-host cell association was observed. The action of PLD could be blocked by the presence of a competitive substrate, phosphatidylethanolamine, during enzyme treatment. The enhancing effect of PLD treatment of the parasites was relatively long lasting since it was still seen 3 h after the enzyme had been removed. The enhancing effect of PLD probably reflected an increased capacity of T. cruzi to associate with host cells rather than increased phagocytosis of PLD-altered parasites by macrophages since similar results were obtained when rat heart myoblasts, which are not phagocytic, were used as host cells. Neither the presence of phospholipids or PLD phospholipid cleavage products during the incubation of T. cruzi with macrophages had any effect on parasite-host cell association. These results show that PLD-mediated alterations to parasite phospholipids increase parasite-host cell association, and suggest that these phospholipids play a role in the initial stages of host cell infection by T. cruzi.

Changes in Trypanosoma cruzi infectivity by treatments that affect calcium ion levels.

The possible role of the intracellular Ca2+ level in the regulation of Trypanosoma cruzi infectivity was explored by measuring the capacity of trypomastigote forms of this organism to invade mammalian host cells after treatments which decrease or elevate cytoplasmic Ca2+. Parasites loaded with either bis-(o-aminophenoxy)-ethane-N,N,N',N' tetraacetic acid (BAPTA) or 2-([2-bis(carboxymethyl)-amino-5-methylphenoxy]methyl)-6-nethoxy-8 - bis(carboxymethyl)aminoquinoline (Quin-2) to chelate Ca2+ displayed significantly decreased infectivity. This effect was denoted by reductions in both the proportion of rat heart myoblasts invaded by the parasite in vitro and the number of trypanosomes penetrating these host cells, the extents of which were BAPTA or Quin-2 concentration dependent. Consistent with these observations, inhibitory effects were also recorded when the parasite was pretreated with the calmodulin-binding phenothiazines trifluoperazine and chlorpromazine or with felodipine, a chemically different type of calmodulin antagonist, for as little as 5 min. In contrast, pretreatment with the Ca2+ ionophore ionomycin, which elevated Ca2+ levels in T. cruzi, significantly enhanced the infective capacity of the parasite. These results point to the existence of a Ca(2+)-dependent mechanism that regulates the invasive capacity of T. cruzi.

The Trypanosoma cruzi immunosuppressive factor (TIF) targets a lymphocyte activation event subsequent to increased intracellular calcium ion concentration and translocation of protein kinase C but previous to cyclin D2 and cdk4 mRNA accumulation.

Many immunosuppressive effects of Trypanosoma cruzi can be reproduced in vitro by a preparation consisting of molecules spontaneously released by this protozoan (termed trypanosomal immunosuppressive factor (TIF)). In this work, we attempted to establish whether TIF-induced inhibition of lymphoproliferation results from preventing lymphocyte activation or impairing a post-activation process. Although [3H]thymidine uptake and expression of CD25 by normal human T lymphocytes stimulated with a phorbol ester were markedly reduced by T. cruzi or TIF, translocation of cytosolic protein kinase C (PKC) to the cell membrane was not affected. Lymphoproliferation induced by ionomycin was also inhibited by T. cruzi or TIF but the typical elevation of intracellular calcium ions [Ca2+]i caused by this calcium ionophore was not altered. The increase in [Ca2+]i induced with anti-CD3 antibody was also unaffected by TIF. TIF did not preclude lymphocytes stimulated with phytohemagglutinin from accumulating normal mRNA levels of NFAT1 (also known as NFATp) and NFATc. NFAT1 and NFATc are components of the NFAT complex that controls transcription of genes coding for several cytokines and whose translocation to the nucleus is dependent upon PKC activation and increased [Ca2+]i. In contrast, the mRNA levels of cyclin D2 and cdk4, which form a holoenzyme complex known to regulate cell progression through the G1 phase, were markedly reduced by TIF. These results indicated that TIF did not inhibit lymphocyte activation leading to early secondary signaling but curtailed a mechanism controlling cell progression through G1 and necessary for reaching S phase.

Knockout of the glutamate dehydrogenase gene in bloodstream Trypanosoma brucei in culture has no effect on editing of mitochondrial mRNAs.

Glutamate dehydrogenase (GDH) was shown previously to bind the 3' oligo[U] tail of the mitochondrial guide RNAs (gRNAs) of Leishmania tarentolae, apparently in the dinucleotide pocket (Bringaud F, Stripecke R, Frech GC, Freedland S, Turck C, Byrne EM, Simpson L. Mol. Cell. Biol. 1997; 17:3915-3923). Bloodstream Trypanosoma brucei cells in culture represent a good system to investigate the genetic effects of knocking out kinetoplastid nuclear genes to test a role in RNA editing, since editing of several mitochondrial genes occurs but is dispensable for viability (Corell RA, Myler P, Stuart K. Mol. Biochem. Parasitol. 1994; 64:65-74 and Stuart K. In: Benne R, editor. RNA editing--the alteration of protein coding sequences of RNA. New York: Ellis Horwood, 1993:25-52). Both GDH alleles of bloodstream T. brucei in culture were replaced by drug resistant markers without any effect on viability. The ratios of edited to unedited mRNAs for several cryptogenes were assayed by primer extension analysis. The steady state abundances of these edited RNAs were unaffected by the double knockout. This evidence suggests that GDH may not play a role in the editing reaction in bloodstream trypanosomes in culture, but this conclusion is tentative since there could be redundant genes for any biological function. We employed a double allelic replacement technique to generate a tetracycline inducible conditional expression of an ectopic copy of the deleted gene in bloodstream trypanosomes in culture. We used this strategy for genes encoding mitochondrial proteins which are not required during this stage of the life cycle, but as a general strategy it should be appropriate for generation of conditional null mutants for essential genes as well.

Cytotoxic effects of normal sera on lymphoid cells. II. Requirements for inhibition of nonspecific serum cytotoxicity by agarose.

Agarose is known to inhibit nonspecifically the cytotoxic effects of normal sera on xenogeneic lymphoid cells. To find an explanation for this agarose effect we have studied its requirements using guinea pig and human sera as the source of activity and rat thymocytes as target cells. Control assays were performed using heat-inactivated (56 C, 30 min) normal rat serum. The inhibitory effect of agarose was readily reproduced with untreated sera and also when sodium ethyleneglycoltetraacetate, a selective chelator of calcium ions, was added to the sera together with excess magnesium. However, the agarose effect failed to occur in the presence of 0.01 M EDTA unless magnesium ions were restored. Abrogation of cytotoxicity in human serum by incubation with a large number of target cells instead of agarose was also found to be magnesium dependent. Titrations of human serum, performed after absorption with agarose in the presence of EDTA, which does not interfere with antigen-antibody binding, and subsequent restoration of divalent cations, revealed no significant change in its cytotoxic titer when compared with that of mock-absorbed serum not subjected to the agarose treatment. Incubation of human serum with either agarose or rat thymocytes resulted in the conversion of factor B, essential for complement activation via the alternative pathway, previously shown to provide the complement activity necessary for the cytotoxic reaction. These results suggest that the agarose effect is mainly attributable to complement consumption via the alternative pathway rather than to the absorption of "natural" antibodies.

Down-regulation of human B lymphocyte activities by a Trypanosoma cruzi membrane glycoprotein.

The effects of purified AGC10, a Trypanosoma cruzi membrane glycoprotein, on normal human B lymphocytes were studied in this work. In the presence of AGC10, [3H]-thymidine uptake by human peripheral blood mononuclear cells stimulated with the B cell-specific mitogen SACI (killed Staphylococcus aureus Cowan I) was markedly decreased. This alteration was accompanied by others such as decreased expression of the CD122 and CD132 chains of the IL-2R complex. These inhibitory effects appeared to be somewhat selective, as expression of CD25, another IL-2R chain, was not affected by AGC10 and no significant modification occurred in the expression of the B-cell-specific marker CD19 or CD21. In contrast, AGC10 did reduce the levels of expression of CD86 and CD80, molecules known to play critical roles in B cell interactions with T lymphocytes. Fairly large subpopulations of, but not all, B lymphocytes had their expression of CD122(+), CD132(+), CD86(+) and CD80(+) reduced to undetectable levels in the presence of AGC10. However, the SACI-activated B cells that remained capable of expressing these molecules in the presence of AGC10 did so at normal levels. This was denoted by comparable mean fluorescence intensity values representing the expression of CD122, CD132, CD86 or CD80 molecules on the surface of SACI-stimulated CD19(+) cells cultured without or with AGC10. These results indicated that AGC10, derived from an organism that causes immunosuppression in infected hosts, down-regulates B cell activities and suggested that the relevant mechanism could involve the molecular alterations described above.

Inhibition by Trypanosoma cruzi of interferon-gamma production by mitogen-stimulated mouse spleen cells.

Infection by Trypanosoma cruzi is accompanied by severe immunosuppression during the acute period. As part of our studies, to define the alterations caused by Trypanosoma cruzi in lymphocyte function, we examined in this work the interferon-gamma (IFN-gamma)-producing capacity of mitogen-stimulated mouse spleen and human peripheral blood mononuclear cells in the presence or absence of blood forms of the parasite. Co-culture of phytohaemagglutinin- or concanavalin A-stimulated spleen cells from normal mice with T. cruzi significantly decreased the levels of IFN-gamma activity found in the supernatants at 48 or 72 h. In contrast, human peripheral blood mononuclear cells, though suppressed by T. cruzi in their capacity to proliferate upon mitogenic stimulation, showed no significant decrease in IFN-gamma production. The addition of exogenous IFN-gamma did not reverse the suppressive effect of T. cruzi on either mouse or human cells. These results revealed, for the first time, the ability of T. cruzi to impair IFN-gamma production by activated mouse lymphocytes. The lack of restoration by exogenous IFN-gamma suggested that the reduced levels of this lymphokine were not, at least by themselves, the causative factor of reduced lymphoproliferation.

Antibody-dependent killing of bloodstream forms of Trypanosoma cruzi by human peripheral blood leukocytes.

Bloodstream forms of Trypanosoma cruzi were found to be destroyed by human lymphoid cells, neutrophils, or eosinophils in the presence of specific antibodies. Nonspecific immunoglobulins present in normal serum did not mediate the cytotoxic reaction. Cells or antibody had no significant lytic effect on the parasite when tested separately and purified, non-activated human adherent cells were inactive antibody was present or not.

Immunization against a challenge with insect vector, metacyclic forms of Trypanosoma cruzi simulating a natural infection.

The protective effects of immunization with an antigen derived from epimastigote forms of Trypanosoma cruzi against challenges with vector metacyclic or bloodstream forms of the parasite were investigated. A marked degree of protection was observed in the immunized mice after challenged with bloodstream or insect-transmissible metacyclic forms. High rates of survival in the immunized groups were accompanied by relatively low, shortlasting parasitemias in some of the animals and significant percentages of immunized mice never developed a measurable parasitemia during the course of the experiments. In contrast, all of the non-immunized animals showed high levels of parasitemias. Similar results were obtained whether the metacyclic challenge was by the intraperitoneal or by the ocular route for which conditions mimicking a natural infection were selected. These results emphasize the protection that immunization confers against challenge with insect-transmissible forms of T. cruzi, and the feasibility of protecting a highly susceptible host against an otherwise lethal acute infection similar to one occurring naturally.

Increased serum levels of an interferon-like activity during the acute period of experimental infection with different strains of Trypanosoma cruzi.

A marked development of an interferon-like antiviral activity was observed in the serum of mice infected with Tulahuén strain Trypanosoma cruzi as early as 24 hours after infection with 500 parasites. Such activity remained elevated until 48 hours postinfection and was reduced to undetectable levels--characteristic of normal, uninfected animals--by 72 hours. Infection with 10(4) Tulahuén strain T. cruzi resulted in production of somewhat lesser levels of interferon activity detectable 12 and 24 hours postinfection but not after 48 hours. Infection with 500 parasites of the Y strain was also followed by an increased interferon-like activity but this was not detectable until day 2 postinfection. During the chronic phase of the disease, interferon-like activity levels were indistinguishable from those of uninfected animals. The possibility that interferon activity may have a regulatory effect on the course of the infection is discussed.

Insect-borne and culture-derived metacyclic Trypanosoma cruzi: differences in infectivity and virulence.

We report in this paper significant differences in the virulence of insect-derived and cultured metacyclic forms of Trypanosoma cruzi which are morphologically indistinguishable. Mice infected intraperitoneally with 10(3) metacyclic T. cruzi isolated from Rhodnius prolixus showed average parasitemia levels greater than 2 X 10(5) organisms/ml around day 10 post-infection (when first measured) and peak levels recorded on day 16 post-infection exceeded 4 X 10(7) organisms/ml. None of these animals survived after 30 days post-infection. In contrast, in mice infected with 10(3) or 10(4) metacyclic forms from axenic cultures the highest average parasitemia was approximately 10(4) organisms/ml and occurred around day 19 post-infection. In these animals, parasitemias declined with time to become undetectable and no mortality was recorded over the 100-day observation period. There was also a marked difference in the 50% lethal dose of insect- and culture-derived metacyclics. The value for the former was 670 parasites whereas none of the mice infected intraperitoneally with up to 10(6) cultured metacyclics died. These results point to a marked difference in the biological properties of insect-borne and cultured T. cruzi metacyclics under our experimental conditions and caution against extending results obtained with the latter to vector-transmissible metacyclics, at least in infectivity and virulence studies.

Evaluation of lymphocyte responsiveness to polyclonal activators during acute and chronic experimental Trypanosoma cruzi infection.

The responses of spleen cells from mice infected with Trypanosoma cruzi to T and B cell-specific mitogens were monitored during the acute and chronic stages of the infection. Responses to either T (phytohemagglutinin or concanavalin A) or B (endotoxic lipopolysaccharide) cell mitogens measured on days 5, 10, 15, and 20 postinfection, i.e., at different times during the acute period, were markedly reduced. Responses measured on days 54 and 90, i.e., during the chronic stage, did not differ significantly from those of normal mouse spleen cells. Proportions of T and B lymphocytes in the spleen were reduced and unaltered, respectively, during acute T. cruzi infection but were comparable to normal values during the chronic state. These results highlight a return of normal T and B lymphocyte responses during chronic experimental Chagas' disease and suggest that transition from the acute to the chronic phase of the infection may be immunologically regulated.