E.L.D. Isola

TRYPANOSOMA CRUZI DIFFERENTIATION AFTER INTERACTION OF EPIMASTIGOTES AND TRIATOMA INFESTANS INTESTINAL HOMOGENATE

Incubation of Trypanosoma cruzi epimastigotes with Triatoma infestans intestinal homogenate leads to differentiation to the metacyclic trypomastigote. Features of this interaction are presented. The morphogenetic mechanism was triggered almost at once; for the minimum interaction period assayed (15 min), the degree of differentiation achieved in Grace medium by Day 6 was 70.0 +/- 9.0%. Longer interaction periods failed to improve differentiation. The morphogenesis became irreversible at 4 hr after interaction. Epimastigotes incubated for 4 hr with T. infestans intestinal homogenate and then washed reached significant differentiation, while those washed before this time failed to do so. Treatment of epimastigotes with albumin improved the experimental conditions thereby hastening morphogenesis, the same percentage of metacyclics occurring in only 4 days. The factors capable of triggering differentiation were adsorbed by T. cruzi epimastigotes, as expected, but also by Leishmania mexicana and, to a lesser degree, by sheep red blood cells. Once the morphogenetic mechanism had been triggered following interaction of epimastigotes with intestinal homogenate for 15 min, metacyclic forms developed when parasites were transferred to Grace but not to other media. Treatment of epimastigotes with trypsin abolished their capacity to differentiate, which was completely reversed following a 5 hr incubation in LIT medium.

Free fatty acids induce cell differentiation to infective forms in Trypanosoma cruzi.

Intestinal extracts of Triatoma infestans induce cell differentiation of Trypanosoma cruzi epimastigotes into the infective metacyclic form. Part of this effect can be explained by the presence of haemoglobin fragments, which stimulate trypanosomal adenylate cyclase. In this work we examined the metacyclogenic activity of lipids present in this intestinal extract. We found that lipid extracts of the intestinal extract have significant stimulatory effects that reside with the free-fatty-acid fraction, especially oleic acid. These compounds stimulate de novo diacylglycerol formation and protein kinase C activity in the parasite. Moreover, metacyclogenesis is stimulated by phorbol esters and cell-permeant diacylglycerol, while protein kinase C down-regulation or incubation with inhibitors of this kinase abrogates this effect. These results indicate that free fatty acids are a novel signal, inducing metacyclogenesis, acting through a pathway involving diacylglycerol biosynthesis and protein kinase C activation.

Central role of extracellular signal-regulated kinase and Toll-like receptor 4 in IL-10 production in regulatory dendritic cells induced by Trypanosoma cruzi

Several Trypanosoma cruzi molecules that stimulate macrophages activity were described as Toll-like receptor 2 (TLR2) ligands. Besides, the models of dendritic cells (DC) are poorly characterised. We have previously demonstrated that live-trypomastigotes (Tp) plus lipopolysaccharide (LPS) induce DC with tolerogenic properties that produce high levels of interleukin (IL)-10 and an impaired capacity to induce lymphoproliferation. Here, we show that the regulatory phenotype was observed with heat-killed trypomastigotes (Tphk) stimulation, ruling out DC infection. T. cruzi induced a particular DC activation state increasing LPS-activation of extracellular regulated kinase (ERK) 1/2 and signal transducer and activator of transcription (STAT) 3. Inhibition of ERK down-regulated IL-10 production and restored DC stimulatory capacity, showing the importance of this pathway in the DC modulation. A recent work shows that signalling via TLR4 and TLR2 induces a synergism in anti-inflammatory cytokine production in murine DC. Upon TLR2 and TLR4 stimulation using Pam(3)Cys or LPS and Tphk in DC from TLR2 knock out (KO) or TLR4-mutant mice, we showed that high levels of IL-10 were independent of TLR2 but associated with TLR4 and NF-kappaB signallization. Although sialic acid has been described as a molecule responsible of DC inhibition, we determine that it is not associated with T. cruzi-IL-10 modulatory response. In conclusion, all these findings demonstrate a key role of ERK and TLR4 in association with NF-kappaB in IL-10 modulation induced by T. cruzi and suggest that this regulatory effect involves parasite-DC interactions not described yet.

Phospholipase A1: A novel virulence factor in Trypanosoma cruzi

Phospholipase A1 (PLA1) has been described in the infective stages of Trypanosoma cruzi as a membrane-bound/secreted enzyme that significantly modified host cell lipid profile with generation of second lipid messengers and concomitant activation of protein kinase C. In the present work we determined higher levels of PLA1 expression in the infective amastigotes and trypomastigotes than in the non-infective epimastigotes of lethal RA strain. In addition, we found similar expression patterns but distinct PLA1 activity levels in bloodstream trypomastigotes from Cvd and RA (lethal) and K98 (non-lethal) T. cruzi strains, obtained at their corresponding parasitemia peaks. This fact was likely due to the presence of different levels of anti-T. cruzi PLA1 antibodies in sera of infected mice, that modulated the enzyme activity. Moreover, these antibodies significantly reduced in vitro parasite invasion indicating the participation of T. cruzi PLA1 in the early events of parasite-host cell interaction. We also demonstrated the presence of lysophospholipase activity in live infective stages that could account for self-protection against the toxic lysophospholipids generated by T. cruzi PLA1 action. At the genome level, we identified at least eight putative genes that codify for T. cruzi PLA1 with high amino acid sequence variability in their amino and carboxy-terminal regions; a putative PLA1 selected gene was cloned and expressed as a recombinant protein that possessed PLA1 activity. Collectively, the results presented here point out at T. cruzi PLA1 as a novel virulence factor implicated in parasite invasion.

Trypanosoma cruzi morphogenesis: preliminary purification of an active fraction from hemolymph and intestinal homogenate of Triatoma infestans.

Differentiation of the Trypanosoma cruzi epimastigote to metacyclic form (Mtc), the parasitic stage in insect vector known to possess infective capacity, has been achieved in vitro by several authors. Insect extracts of Rhodnius prolixus (Wood and Souza, 1976, Revista do Instituto de Medicina Tropical do Sao Paulo 18: 93-96), Triatoma infestans (Isola et al., 1981, Journal of Parasitology 67: 53-58; 1983, Revista Argentina de Microbiologia 15: 181-185), or Phylosamia cynthia (Wood and Pipkin, 1969, Experimental Parasitology 24: 176-183), as well as T. infestans embryo cells (Lanar, 1979, Journal of Protozoology 26: 457-462), have been used as culture supplement. The in vitro activity of fractions obtained from hemolymph and intestinal homogenate of T. infestans, one of the most widespread vectors of this parasite, was analyzed in an attempt to determine the active components involved in the differentiation process. Epimastigotes of the Tulahuen isolate of T. cruzi maintained by weekly passages in biphasic medium were used to sow the culture media for differentiation assays. Intestinal homogenate and hemolymph of T. infestans were obtained as already reported (Isola et al., 1981, 1983, loc. cit.). Hindguts were removed and macerated in Grace medium to supplement cultures or in 0.15 M phosphate buffered saline (PBS), pH 7.2, when used for gel filtrations, in a ratio of 2.5-3.0 hindguts/ml. The suspensions were centrifuged for 30 min at 12,000 g, sterilized by filtration through 0.2 ,m pores, and kept at -70 C. Three to 5 ml of either substrate (hemolymph or intestinal homogenate) were filtered through a 100 x 2.6 cm Sephadex G-200 column at 4 C or 20-22 C, eluted with PBS 0.07 M, pH 7.2, and read at 280 nm (Dextran Blue 600K, human IgG fluorescein labelled 200K, human albumin 67K, ovoalbumin 43K, myoglobine 17.8K, and ribonuclease 13.7K were used as references). The fractions recovered were sterilized through 0.2 Aum pores, lyophilized and resuspended in Grace medium up to similar protein concentration in each fraction ranging from 0.4 to 0.7 mg/ml in different experiments. Samples of both substrates were also filtered through a 100 x 1.5 cm Sephadex G-50 column at 20-22 C, keeping elution and the fractioning process as mentioned above. The protein concentration of intestinal homogenate, hemolymph, and the fractions was determined by the method reported by Lowry et al. (1951, Journal of Biological Chemistry 193: 265-275). The protein level of intestinal homogenate was evaluated in PBS batches and a similar hindgut/ml ratio (2.5-3.0/ml) was maintained when the homogenate was prepared in Grace medium. The differentiation assays were carried out in Grace medium modified by Junker et al. (1967, Science 155: 1565-1566) (Gibco Laboratories Life Technology Inc.), Grace medium supplemented with intestinal homogenate (Isola et al., 1981, loc. cit.), Grace medium supplemented with 2% v/v hemolymph of T. infestans (Isola et al., 1983, loc. cit.), and GFx medium, prepared by supplementing Grace medium with 1 of the fractions (Fx) obtained by gel filtration (Fx-200 or Fx-50, depending on the Sephadex pore size). Each experimental variable was assayed in quintuplicate. Cultures were sown with 4 x 106 epimastigotes/ml and the percentage of differentiation to the Mtc stage was determined in at least 200 forms in wet and fixed-stained (May Griinwald-Giemsa) preparations. Cultures were examined every 48 hr during 15 days, and morphogenesis was evaluated as already reported (Isola et al., 1981, loc. cit.). The optimal protein concentration to induce Mtc differentiation was determined in Grace medium supplemented with 0.5 to 4.0 mg/ml of intestinal homogenate, as well as 0.12 to 0.50 mg/ml of the F2-50 of the same substrate in day 7. The F2-50 of intestinal homogenate was con-

Phospholipases A in Trypanosomatids

Phospholipases are a complex and important group of enzymes widespread in nature, that play crucial roles in diverse biochemical processes and are classified as A1, A2, C, and D. Phospholipases A1 and A2 activities have been linked to pathogenesis in various microorganisms, and particularly in pathogenic protozoa they have been implicated in cell invasion. Kinetoplastids are a group of flagellated protozoa, including extra- and intracellular parasites that cause severe disease in humans and animals. In the present paper, we will mainly focus on the three most important kinetoplastid human pathogens, Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp., giving a perspective of the research done up to now regarding biochemical, biological, and molecular characteristics of Phospholipases A1 and A2 and their contribution to pathogenesis.

Lipids from attenuated and virulent Babesia bovis strains induce differential TLR2-mediated macrophage activation.

Babesia bovis is an intraerythrocytic apicomplexan protozoa of cattle that causes an acute infection with parasite persistence. Babesiosis limitation depends on macrophages, essential effector cells of the host innate defense, which generate inflammatory cytokines and nitric oxide. Herein, we report quantitative differences in the lipid composition of merozoites from two B. bovis strains with polar behaviour: attenuated R1A and virulent S2P. Accordingly, we observed a distinct inflammatory response induced by the total lipids of R1A (L(A)) and S2P (L(V)) in murine peritoneal macrophages. L(A) and particularly its fractions phosphatidic acid and phosphatidylserine+phosphatidylinositol (PS+PI), produced a strong activation of these cells with lipid body formation, cyclooxygenase-2 expression and pro-inflammatory TNFalpha, IL-6 and KC secretion. Although L(V) did not activate these cells, the corresponding PS+PI fraction induced TNFalpha, IL-6 and KC release. Therefore, these facts might be suggesting the presence of an inhibitor in L(V). Furthermore, the employment of wild type and toll like receptor 2 knockout (TLR2KO) mice allowed us to demonstrate that macrophage activation by the stimulating lipid fractions was mediated through TLR2. Interestingly, only L(A) activated the extracellular signal-regulated kinases 1 and 2 (ERK1/2). Inhibitory studies employing UO126, indicated that the ERK pathway was required for TNFalpha, IL-6 and KC release. In conclusion, the absence of inflammatory response observed with the lipids of S2P virulent strain could constitute an evasion mechanism of the innate immune response enabling parasite establishment in the host.

Trypanosoma cruzi: Differentiation to metacyclic trypomastigotes in the presence of ADP-ribosyltransferase inhibitors

The participation of ADP-ribosyltransferase in Trypanosoma cruzi differentiation to the metacyclic stage was evaluated by analyzing morphogenesis blockage by specific enzyme inhibitors: benzamide, 3-aminobenzamide, theophylline, and nicotinamide. In vitro assays showed a statistically significant reduction in the number of metacyclic forms only when any one of the four inhibitors was added during the period of interaction between epimastigote and Triatoma infestans intestinal homogenate or when present throughout the subsequent culture period in Graces medium. When nicotinamide or benzamide was present during both interaction and culture period, morphogenesis was virtually abolished (less than or equal to 2%). In the in vivo assays, mice inoculated with parasites obtained from the insect vectors fed with trypomastigote-infected blood containing one of the four enzyme inhibitors developed lower parasitemias and showed longer survival in every case, compared with the respective controls. These findings suggest ADP-ribosyltransferase participation in T. cruzi differentiation both in vitro and in vivo.

Trypanosoma cruzi: inhibition of metacyclogenesis by mannose.

Metacyclogenesis of Trypanosoma cruzi epimastigotes was evaluated in a medium supplemented with Triatoma infestans intestinal homogenate in the presence of sugars and derivates as are mannose, galactose, fucose, N-acetylglucosamine, mannose 6-P, and fructose 1,6-P at a concentration of 25 mM. Only mannose significantly inhibited metacyclogenesis. Sodium metaperiodate and trypsin treatment of the intestinal homogenate also inhibited differentiation. In our opinion there exists a proteinic factor in the intestine of the vector that promotes metacyclogenesis and is incorporated by the parasite. Treatment of the intestinal homogenate with alkaline phosphatase had no effect. Instead, high ionic strength in the medium (0.4 M NaCl) strongly inhibited metacyclogenesis indicating that, in these conditions, the possible binding of the differentiation factor to the parasite surface was inhibited.

Inhibition of Early Endosome Fusion by Trypansom cruzi-Infected Macrophage Cytosol

ABSTRACT. Trypanosoma cruzi trypomastigotes survive inside macrophages by promoting fusion between the parasitophorous vacuole and mature host lysosomes upon internalization. Since trypomastigotes can evade the lytic pathway, the earliest steps of endocytosis, such as early endosome fusion, may be affected. To test this hypothesis, we used an in vitro early endosome fusion assay. Our results show that trypomastigote‐infected macrophage cytosols cannot promote fusion between early endosomes, compared to mock‐infected cytosols (heat‐killed trypomastigotes were used in the parasite‐macrophage interaction assay). GTPγS addition potentiates the fusogenic activity driven by trypomastigote‐infected macrophage cytosol‐mediated assays, unlike the biphasic fusogenic effect obtained with GTPγS treatment of macrophage cytosol controls. Calcium‐stimulated early endosome fusogenic processes are not affected in the assays mediated by infected macrophage cytosol. We conclude that GTP‐regulated factors, and not calcium‐regulated elements, are involved in the inhibition of the early endosome fusogenic process by the trypomastigote‐infected macrophage cytosol. This primary impediment to the progress of a normal endocytosis may be a relevant step required for the lysosomal recruitment‐fusion of the host lysosmes upon trypomastigote infection and further survival of the parasite within its host.