scholarly journals Autoimmunity to phosphatidylserine and anemia in African Trypanosome infections

2021 ◽  
Vol 15 (9) ◽  
pp. e0009814
Author(s):  
Juan Rivera-Correa ◽  
Joseph Verdi ◽  
Julian Sherman ◽  
Jeremy M. Sternberg ◽  
Jayne Raper ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
Trypanosoma Cruzi ◽  
Trypanosoma Brucei ◽  
Mouse Models ◽  
Human African Trypanosomiasis ◽  
Annexin V ◽  
Trypanosome Infection ◽  
Erythrocyte Plasma Membrane

Anemia caused by trypanosome infection is poorly understood. Autoimmunity during Trypanosoma brucei infection was proposed to have a role during anemia, but the mechanisms involved during this pathology have not been elucidated. In mouse models and human patients infected with malaria parasites, atypical B-cells promote anemia through the secretion of autoimmune anti-phosphatidylserine (anti-PS) antibodies that bind to uninfected erythrocytes and facilitate their clearance. Using mouse models of two trypanosome infections, Trypanosoma brucei and Trypanosoma cruzi, we assessed levels of autoantibodies and anemia. Our results indicate that acute T. brucei infection, but not T. cruzi, leads to early increased levels of plasma autoantibodies against different auto antigens tested (PS, DNA and erythrocyte lysate) and expansion of atypical B cells (ABCs) that secrete these autoantibodies. In vitro studies confirmed that a lysate of T. brucei, but not T. cruzi, could directly promote the expansion of these ABCs. PS exposure on erythrocyte plasma membrane seems to be an important contributor to anemia by delaying erythrocyte recovery since treatment with an agent that prevents binding to it (Annexin V) ameliorated anemia in T. brucei-infected mice. Analysis of the plasma of patients with human African trypanosomiasis (HAT) revealed high levels of anti-PS antibodies that correlated with anemia. Altogether these results suggest a relation between autoimmunity against PS and anemia in both mice and patients infected with T. brucei.

scholarly journals Differential Inhibition of Human and Trypanosome Ubiquitin E1S by TAK-243 Offers Possibilities for Parasite Selective Inhibitors

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
D. Roeland Boer ◽  
Marie-José Bijlmakers
Keyword(s):  
Amino Acids ◽  
Trypanosoma Cruzi ◽  
Trypanosoma Brucei ◽  
Human African Trypanosomiasis ◽  
Potent Inhibitor ◽  
Model System ◽  
Differential Inhibition ◽  
Protein Ubiquitination ◽  
Related Compounds

Abstract Novel strategies to target Trypanosoma brucei, Trypanosoma cruzi and Leishmania are urgently needed to generate better and safer drugs against Human African Trypanosomiasis, Chagas disease and Leishmaniasis, respectively. Here, we investigated the feasibility of selectively targeting in trypanosomatids the ubiquitin E1 activating enzyme (UBA1), an essential eukaryotic protein required for protein ubiquitination. Trypanosomatids contain two UBA1 genes in contrast to mammals and yeast that only have one, and using T. brucei as a model system, we show that both are active in vitro. Surprisingly, neither protein is inhibited by TAK-243, a potent inhibitor of human UBA1. This resistance stems from differences with the human protein at key amino acids, which includes a residue termed the gatekeeper because its mutation in E1s leads to resistance to TAK-243 and related compounds. Importantly, our results predict that trypanosomatid selective UBA1 inhibition is feasible and suggest ways to design novel compounds to achieve this.

scholarly journals Anti-Trypanosomal Proteasome Inhibitors Cure Hemolymphatic and Meningoencephalic Murine Infection Models of African Trypanosomiasis

2020 ◽  
Vol 5 (1) ◽  
pp. 28 ◽  
Author(s):  
Srinivasa P S Rao ◽  
Suresh B Lakshminarayana ◽  
Jan Jiricek ◽  
Marcel Kaiser ◽  
Ryan Ritchie ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Mouse Models ◽  
Human African Trypanosomiasis ◽  
Proteasome Inhibitors ◽  
African Trypanosomiasis ◽  
Single Digit ◽  
Pharmacological Characterization ◽  
Brain Infection

Current anti-trypanosomal therapies suffer from problems of longer treatment duration, toxicity and inadequate efficacy, hence there is a need for safer, more efficacious and ‘easy to use’ oral drugs. Previously, we reported the discovery of the triazolopyrimidine (TP) class as selective kinetoplastid proteasome inhibitors with in vivo efficacy in mouse models of leishmaniasis, Chagas Disease and African trypanosomiasis (HAT). For the treatment of HAT, development compounds need to have excellent penetration to the brain to cure the meningoencephalic stage of the disease. Here we describe detailed biological and pharmacological characterization of triazolopyrimidine compounds in HAT specific assays. The TP class of compounds showed single digit nanomolar potency against Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense strains. These compounds are trypanocidal with concentration-time dependent kill and achieved relapse-free cure in vitro. Two compounds, GNF6702 and a new analog NITD689, showed favorable in vivo pharmacokinetics and significant brain penetration, which enabled oral dosing. They also achieved complete cure in both hemolymphatic (blood) and meningoencephalic (brain) infection of human African trypanosomiasis mouse models. Mode of action studies on this series confirmed the 20S proteasome as the target in T. brucei. These proteasome inhibitors have the potential for further development into promising new treatment for human African trypanosomiasis.

scholarly journals Cross-Resistance to Nitro Drugs and Implications for Treatment of Human African Trypanosomiasis

2010 ◽  
Vol 54 (7) ◽  
pp. 2893-2900 ◽  
Author(s):  
Antoaneta Y. Sokolova ◽  
Susan Wyllie ◽  
Stephen Patterson ◽  
Sandra L. Oza ◽  
Kevin D. Read ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Human African Trypanosomiasis ◽  
Parent Drug ◽  
Cross Resistance ◽  
Pharmacokinetic Studies ◽  
African Trypanosomiasis ◽  
Trypanosoma Brucei Brucei ◽  
Resistant Lines

ABSTRACT The success of nifurtimox-eflornithine combination therapy (NECT) for the treatment of human African trypanosomiasis (HAT) has renewed interest in the potential of nitro drugs as chemotherapeutics. In order to study the implications of the more widespread use of nitro drugs against these parasites, we examined the in vivo and in vitro resistance potentials of nifurtimox and fexinidazole and its metabolites. Following selection in vitro by exposure to increasing concentrations of nifurtimox, Trypanosoma brucei brucei nifurtimox-resistant clones designated NfxR1 and NfxR2 were generated. Both cell lines were found to be 8-fold less sensitive to nifurtimox than parental cells and demonstrated cross-resistance to a number of other nitro drugs, most notably the clinical trial candidate fexinidazole (∼27-fold more resistant than parental cells). Studies of mice confirmed that the generation of nifurtimox resistance in these parasites did not compromise virulence, and NfxR1 remained resistant to both nifurtimox and fexinidazole in vivo. In the case of fexinidazole, drug metabolism and pharmacokinetic studies indicate that the parent drug is rapidly metabolized to the sulfoxide and sulfone form of this compound. These metabolites retained trypanocidal activity but were less effective in nifurtimox-resistant lines. Significantly, trypanosomes selected for resistance to fexinidazole were 10-fold more resistant to nifurtimox than parental cells. This reciprocal cross-resistance has important implications for the therapeutic use of nifurtimox in a clinical setting and highlights a potential danger in the use of fexinidazole as a monotherapy.

scholarly journals A Novel BCL2-Driven Compound Mutant Mouse Model for In Vivo Research on BH3 Mimetics

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 39-39
Author(s):  
Ismini Halmer ◽  
Alexandra da Palma Guerreiro ◽  
Laura Beckmann ◽  
Christian Reinhardt ◽  
Hamid Kashkar ◽  
...  
Keyword(s):  
Overall Survival ◽  
B Cells ◽  
Mouse Model ◽  
Mouse Models ◽  
Clinical Symptoms ◽  
Conflicts Of Interest ◽  
Bh3 Mimetics ◽  
Normal Ranges

Introduction: Eµ-TCL1-transgenic mouse models are often applied to discover and observe the development and kinetic of chronic lymphocytic leukaemia (CLL), as they develop diseases most similar to human CLL with a very high penetrance. To gain a better understanding on new therapy options and their effect on disease regression it is very important to observe therapy response, overall survival and symptoms during treatment of the disease not only in vitro but also in vivo in a suitable mouse model. However, application of BH3 mimetics like venetoclax is limited in the classical Eµ-TCL1 mouse model, since these mice are resistant towards venetoclax treatment. Therefore, we have generated a novel mouse model with Eµ-TCL1 as back bone and conditional overexpression of BCL2. Methods and results: We established a new mouse model (TBC) by crossbreeding mice expressing Eµ-TCL1tg/wtwith mice containing a B-cell specific conditional Bcl-2Rosa26/wt; Cd19CreCre/wtoverexpression and compared the disease kinetics to classical Eµ-TCL1 mice and to BC mice. TBC animals exhibit a severe leukocytosis at very early stages of disease development (12 weeks; mean 96.000/µl) in comparison to TC (15.100/µl) and BC (81.900/µl) mice. TBC mice develop CD23low/CD21neg leukemic B cells as they are known from TC mice with CD19+/CD5+ expression. Indeed, these mice show a significantly shortened overall survival of ~300 days (n=43) compared to TC mice (n=106; ~350 days; p<0.001) and BC mice (n=28; ~410 days; p<0.001) with severe clinical symptoms such as splenomegaly and cachexia. Strikingly, in contrast classical TC mice, which are resistant towards venetoclax, isolated B-cells of TBC mice are 10-times more sensitive towards venetoclax in vitro (0,02 µM) and can also be killed by the MCL1 inhibitors in nanomolar ranges, but not by BCL-xl inhibitors (>2µM). Based on our in vitro data, we have treated TBC mice with venetoclax and observed an early and dramatic drop of leukocytes to normal ranges within the first two weeks of treatment. Leukocyte reduction lasted for the whole period of treatment. When investigating the spleens after sacrificing the mice they showed high amounts of dead cells inside the spleens, indicating that venetoclax was also efficient in lymphatic tissues as we know it from human trials. Conclusions: Autochthonous mouse models on which BH3 mimetics can be tested are rare. In our mouse model apoptosis screening in vitro we can show good results for BH3 mimetics with a high sensitivity already in low dosing. The BCL2-driven TCL1 mouse model enables the investigation of treatment with venetoclax in vivo to gain a better understanding of this frequently on patients applied therapy. Moreover, this model will help us to test other drugs (like MCL1 inhibitors) in combination with venetoclax to identify synergistic drugs in vivo in a timely manner. Furthermore, this model will offer us the opportunity to identify treatment strategies to overcome venetoclax resistance in vivo. Disclosures No relevant conflicts of interest to declare.

55 LIVE PLASMA MEMBRANE ANALYSIS OF EARLY APOPTOTIC CELL DEATH IN PORCINE ADULT DERMAL FIBROBLASTS PRIOR TO SOMATIC CELL CLONING

2008 ◽  
Vol 20 (1) ◽  
pp. 108
Author(s):  
M. Skrzyszowska ◽  
M. Samiec
Keyword(s):  
Plasma Membrane ◽  
Somatic Cell ◽  
Dermal Fibroblast ◽  
Chemical Activation ◽  
Annexin V ◽  
Fibroblast Cells ◽  
Apoptotic Cells ◽  
Cell Cloning ◽  
Donor Cells

The aim of our study was to determine the in vitro developmental capability of porcine nuclear-transferred (NT) embryos reconstructed with adult dermal fibroblast cells, which had been analyzed for apoptosis by live plasma membrane fluorescent labelling. Frozen/thawed fibroblasts, which had been in vitro cultured to confluency, were used for analysis. To detect the early apoptotic changes in the plasma membrane involving the externalization of phosphatidylserine molecules and the subsequent loss of lipid composition asymmetry, the fibroblasts were tagged using a conjugate of annexinV with enhanced green fluorescent protein (eGFP). In the somatic cell cloning procedure, enucleated in vitro-matured oocytes were reconstituted with non-apoptotic dermal fibroblast cell nuclei. Afterwards, NT-derived oocytes were stimulated with a combination of electrical and chemical activation. Simultaneous fusion and electrical activation were induced by application of two successive DC pulses of 1.2 kV cm–1 for 60 �s. A two-step chemical activation procedure was initiated after a 1.5–2 h delay. The cybrids were exposed to 15 µm calcium ionomycin for 5 to 7 min and then incubated in the culture medium supplemented with 10 µg mL–1 cycloheximide for 3 h. Reconstructed embryos were in vitro cultured in NCSU-23 medium for 6–7 days. Fluorescence analysis of the adult dermal fibroblast cells revealed that a relatively high proportion of donor cells exhibited proapoptotic changes in the plasma membrane. The percentage of late apoptotic cells with advanced morphological changes did not exceed 30%. Moreover, an extremely low rate (ranging from 0 to 2%) of early apoptotic cells, with a morphologically normal, i.e., smooth (non-corrugated) and intact (non-blebbing), plasmolemma but which emitted the green eGFP-derived chemiluminescence, was detected. A total of 219 enucleated oocytes were subjected to reconstruction and 185/219 (84.5%) were successfully fused with non-apoptotic nuclear donor cells. Out of 185 cultured NT embryos, 108 (58.4%) cleaved. The frequencies of cloned embryos, that reached the morula and blastocyst stages, were 84/185 (45.4%) and 26/185 (14.0%), respectively. In conclusion, annexin V-eGFP is a sensitive method able to detect the early phases of apoptosis in cultured adult dermal fibroblast cells, because it identified that very small proportion of morphologically normal cells (without shrinkage of the plasmolemma) that also emitted the annexin V-eGFP-derived biochemiluminescence. Nonetheless, the probability of their random erroneous selection for somatic cell cloning appears to be extremely low. It was also found that the preimplantation developmental potential of NT embryos originating from non-apoptotic adult dermal fibroblast cells is relatively high. This work was supported by the Scientific Net of Animal Reproduction Biotechnology.

An improved polarized rat hepatoma hybrid cell line. Generation and comparison with its hepatoma relatives and hepatocytes in vivo

1994 ◽  
Vol 107 (4) ◽  
pp. 813-825 ◽  
Author(s):  
M.R. Shanks ◽  
D. Cassio ◽  
O. Lecoq ◽  
A.L. Hubbard
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
Membrane Proteins ◽  
Cell Line ◽  
Hybrid Cell ◽  
The Other ◽  
Hepatocyte Polarity ◽  
Rat Hepatoma

Studies of hepatocyte polarity, an important property of liver epithelial cells, have been hampered by the lack of valid in vitro models. We report here that a new polarized hepatoma-derived hybrid cell line, called WIF-B, has improved characteristics to those of its parent, WIF12-1. This latter line originated from the fusion of non-polarized rat hepatoma Fao cells with human fibroblasts (WI-38) and selection for a polarized phenotype. We generated the WIF-B line by growing WIF12-1 cells as unattached aggregates for three weeks and selecting for survivors. Karyotype analysis showed a broad chromosome pattern in the initial WIF-B population, but this pattern stabilized after a few passages. The growth and phenotypic properties of these cells were quite different from those of their polarized WIF12-1 parent. WIF-B cells attained a 4-fold higher maximal density in monolayer culture, survived at this density for > 5 days rather than 1 day, and exhibited two to three times more apical structures during this period (80 to 95%). We compared several parameters of liver differentiation in the WIF-B cells with those of a related hybrid clone, WIF12-E, which is extinguished for most liver-specific functions, and with the common hepatoma parent, Fao. By immunoblot analysis, the levels of expression of eight plasma membrane proteins were higher in the WIF-B cells than in either of the other two cell lines and ranged from 10 to 200% of those in vivo. Two plasma membrane proteins were not detected in WIF12-E cells. By immunofluorescence, the apical membrane proteins in WIF-B displayed different cellular localizations than in either of the other two cell lines. In WIF-B cells, apical proteins were confined to a plasma membrane region that we have identified as the apical domain by several criteria (Ihrke, G., Neufeld, E.D., Meads, T., Shanks, M.R., Cassio, D., Laurent, M., Schroer, T.A., Pagano, R. E. and Hubbard, A. L. J. Cell Biol., 123, 1761–1765). The same molecules were distributed over the entire plasma membrane of Fao and WIF12-E cells and also (for Fao cells) in intracellular punctate structures that did not colocalize with the majority of structures containing a secretory protein, albumin. Our results indicate that the WIF-B cells are more highly differentiated than any of their ancestors (Fao or WIF12-1 cells) and thus, are promising candidates for in vitro studies of hepatocyte polarity.

BCX-4208 (RO5092888), a Purine Nucleoside Phosphorylase (PNP) Inhibitor, Is a Novel, Potent Orally Active Anti-T-Cell and B-Cell Agent.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1547-1547
Author(s):  
Shanta Bantia ◽  
Cynthia Parker ◽  
Ramanda Upshaw ◽  
John Michael Kilpatrick ◽  
Amanda Cunningham ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Lymphocyte Proliferation ◽  
Clinical Investigation ◽  
Human Lymphocytes ◽  
Annexin V ◽  
Fold Increase ◽  
Cell Immunity

Abstract The profound suppression of T-cell immunity seen in patients with an inherited PNP deficiency supports the potential application of inhibitors of this purine salvage enzyme in the therapy of T-cell malignancies and T-cell mediated autoimmune diseases. About thirty percent of PNP deficient patients also show evidence of B-cell dysfunction. BCX-4208 is a novel potent transition state analog inhibitor of PNP (IC50 ~ 0.0005 μM) and in the presence of 10 μM deoxyguanosine (dGuo), inhibits human lymphocyte proliferation induced by MLR, IL-2 or Con-A with IC50s of 0.159, 0.26 and 0.73 μM, respectively. The IC50 for dGuo in the same assays in the presence of 1 μM BCX-4208 ranges from 1–3 μM. Neither BCX-4208 alone nor dGuo alone inhibits proliferation of lymphocytes. In the presence of PNP inhibitor, dGuo is converted to dGMP and then to dGTP. Accumulation of dGTP results in the alteration of deoxynucleotide (dNTP) pools, causing death of cells via a mechanism characteristic of apoptosis. In vitro data demonstrates that following exposure to BCX-4208 and dGuo, dGTP in human lymphocytes is elevated and a 5–8 fold increase in dGTP results in 50% inhibition of lymphocyte proliferation. Flow cytometric analyses of human lymphocytes using annexin-V staining reveal that BCX-4208 in the presence of dGuo induces cellular apoptosis not only in T cells (CD3+), but also in B cells (CD20+; CD19+) (Table 1). BCX-4208 is orally bioavailable in mice, can achieve maximal inhibition of PNP, and elevates plasma dGuo levels to 3–5 μM (predose levels < 0.004 μM), which is similar to levels seen in PNP deficient patients and to levels needed to cause apoptosis in T and B-cells. These data support the evaluation of BCX-4208 in the treatment of not only T-cell mediated diseases but also B-cell mediated diseases. BCX- 4208 is currently undergoing early clinical investigation in patients with psoriasis. Table 1. Cell subsets % Apoptotic cells mean ± SEM (n = 4–7) Vehicle Treatment group *p ≤ 0.01 compared to vehicle CD3+ 7.2 ± 0.9 18.3 ± 3.7* CD20+ 24.0 ± 3.6 41.8 ± 6.2* CD19+ 25.0 ± 3.1 51.6 ± 7.4*

The Para-Isomer of Nitric Oxide Donating Acetylsalicylic Acid (p-NO-ASA) Induces Apoptosis in Chronic Lymphocytic Leukemia (CLL) in Vitro and In Vivo without Gross Systemic Toxicities.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3783-3783
Author(s):  
Regina Razavi ◽  
Iris Gehrke ◽  
Rajesh Kumar Gandhirajan ◽  
Simon Jonas Poll-Wolbeck ◽  
Julian Paesler ◽  
...  
Keyword(s):  
B Cells ◽  
Tumor Volume ◽  
Annexin V ◽  
Immunoblot Analysis ◽  
Vehicle Control ◽  
Lymphocytic Leukemia ◽  
Atp Content ◽  
Meta Isomer

Abstract Abstract 3783 Poster Board III-719 Chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature, non functional B cells. WNT/β-catenin(CTNNB1)/TCF/Lef-1 signalling appears to be constitutively and aberrantly activated in these cells. Furthermore, several compounds related to the non-steroidal anti-inflammatory drugs (NSAIDs) are reported to inhibit β-catenin stability and/or function in WNT active cancers in vitro. However, so far clinical studies with such substances generated disappointing results which is likely to the fact that therapeutic plasma concentrations could not be reached without producing significant toxicities; hence, the required high concentrations limit their clinical use. Recently, nitric oxide-donating acetylsalicylic acid (NO-ASA) has been shown to achieve high plasma levels in doses not leading to any relevant side effects in humans. In addition, NO-ASA could disrupt complexation of β-catenin and TCF-4 in vitro. Because the general structure of NO-ASA enables more variants, the aim of our study was to evaluate the effect of the para- (p-NO-ASA) and meta-isomer (m-NO-ASA) in CLL in vitro and in vivo. Primary CLL cells as well as healthy peripheral blood monocytes (PBMCs) and healthy B cells were treated with varying concentrations of p- and m-NO-ASA. Cytotoxicity was assessed by microscopic cell viability testing and measurement of the reduction of the ATP content. Induction of apoptosis was investigated by Annexin V-FITC/propidiumiodide staining and immunoblotting of the caspases-9, -3 as well as PARP. Further, the β-catenin protein amount and the expression of WNT effector proteins like cyclin D1 (CCND1), C-MYC and LEF-1 was evaluated by immunoblot analysis. In vivo activity of NO-ASA was evaluated by treating irradiated CD1 nu/nu female mice, containing a JVM-3 cell line xenograft, with 100 mg/kg/day of p- and m-NO-ASA or vehicle control p.o. for 3 weeks continuously. We found a significant concentration dependent reduction of the ATP content in CLL cells after treatment with p-NO-ASA, whereas the meta-isomer showed no effect on CLL cells. While healthy B cells and healthy PBMCs were not significantly affected by any of the isomers the mean lethal concentration (LC50) was 4.64 μM in CLL cells. Annexin V-FITC/PI staining revealed that reduced cell survival occurs in a time and concentration dependent manner and is mediated by apoptosis. Treatment with 10 μM of p-NO-ASA for 24 hours reduced survival to 46.3 ± 10.1%. This effect was achieved as early as 6 hours after treatment. Immunoblot analysis showed that only p-NO-ASA but not m-NO-ASA activates caspases-9, -3 and cleaves PARP at the same concentrations, which lead to induction of cell death. β-catenin protein levels and WNT pathway target genes are down regulated between 1 to 10 μM also only by the para-isomer. In vivo results revealed that exclusively p-NO-ASA show a strong antitumor efficacy with an IRmax value of 83.1%. After 9 days of treatment p-NO-ASA lead to a significantly reduced tumor volume compared to vehicle control (126.4 ± 22.3 mm3 for p-NO-ASA vs. 290.0 ± 65.9 mm3 for the vehicle control, p=0.0303). Tumor volume of vehicle treated controls increased up to 775.4 ± 219.6 mm3 whereas the tumor volume of p-NO-ASA treated group remained stable at 128.7 ± 27.6 mm3 (p=0.0091) over a treatment period of 21 days. The meta-isomer exhibited no significant antineoplastic effect. Our findings show that the para- but not the meta-isomer of NO-ASA selectively induces caspase mediated apoptosis in CLL cells. The mechanism of action might include inhibition of β-catenin/Lef-1 signaling since we observed downregulation of specific target gene expression. Due to our promising in vivo results, discovering a strong inhibition of tumor growth without producing gross side effects, p-NO-ASA might be a valuable compound for the treatment of CLL. More investigations of the mechanism of action and the specific difference between the positional isomers are needed. Disclosures: Hallek: Roche: Consultancy, Honoraria, Research Funding.

scholarly journals Formation and Remodeling of Inositolphosphoceramide during Differentiation of Trypanosoma cruzi from Trypomastigote to Amastigote

2003 ◽  
Vol 2 (4) ◽  
pp. 756-768 ◽  
Author(s):  
Maria Laura Salto ◽  
Laura E. Bertello ◽  
Mauricio Vieira ◽  
Roberto Docampo ◽  
Silvia N. J. Moreno ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Trypanosoma Cruzi ◽  
Palmitic Acid ◽  
Phospholipase C ◽  
Phospholipase A ◽  
Acidic Ph ◽  
Acid Hydrolase ◽  
Aureobasidin A

ABSTRACT Differentiation of Trypanosoma cruzi trypomastigotes to amastigotes inside myoblasts or in vitro, at low extracellular pH, in the presence of [3H]palmitic acid or [3H]inositol revealed differential labeling of inositolphosphoceramide and phosphatidylinositol, suggesting that a remodeling process takes place in both lipids. Using 3H-labeled inositolphosphoceramide and phosphatidylinositol as substrates, we demonstrated the association of at least five enzymatic activities with the membranes of amastigotes and trypomastigotes. These included phospholipase A1, phospholipase A2, inositolphosphoceramide-fatty acid hydrolase, acyltransferase, and a phospholipase C releasing either ceramide or a glycerolipid from the inositolphospholipids. These enzymes may be acting in remodeling reactions leading to the anchor of mature glycoproteins or glycoinositolphospholipids and helping in the transformation of the plasma membrane, a necessary step in the differentiation of slender trypomastigotes to round amastigotes. Synthesis of inositolphosphoceramide and particularly of glycoinositolphospholipids was inhibited by aureobasidin A, a known inhibitor of fungal inositolphosphoceramide synthases. The antibiotic impaired the differentiation of trypomastigotes at acidic pH, as indicated by an increased appearance of intermediate forms and a decreased expression of the Ssp4 glycoprotein, a characteristic marker of amastigote forms. Aureobasidin A was also toxic to differentiating trypomastigotes at acidic pH but not to trypomastigotes maintained at neutral pH. Our data suggest that inositolphosphoceramide is implicated in T. cruzi differentiation and that its metabolism could provide important targets for the development of antiparasitic therapies.

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