Junko Nakajima-Shimada

Inhibition of Fas-mediated apoptosis by Trypanosoma cruzi infection.

Trypanosoma cruzi-infected and normal control mammalian cells were subjected to analysis of Fas-mediated apoptosis stimulated by an agonistic anti-Fas monoclonal antibody. The infected cells showed markedly hampered apoptotic changes in nuclear morphology, phosphatidylethanolamine translocation from the inside to the outside of the plasma membrane, and DNA fragmentation into multiples of 180 bp, relative to normal control cells. Upstream of these morphological and biochemical consequences, the caspase-3 activity was elevated by the Fas stimulation in a significantly greater proportion of intact control cells, but at a highly reduced rate of infected cells. The rapid elevation of caspase-8 activity in control, apoptotic cells was completely inhibited in infected cells. In an examination of the specificity of other stimulants, X-ray radiation or chemicals such as hydrogen peroxide, colchicine or etoposide did not cause significant differences in apoptotic rates between control and infected cells; tumor necrosis factor-alpha, however, induced a high rate of apoptosis in control cells, with an extremely lowered rate in infected cells. This study demonstrates, for the first time, that T. cruzi infection inhibits one of the earliest steps of death receptor-mediated apoptosis, an effect that most probably involves the inhibition of caspase-8. Differential apoptotic responses in cells infected with T. cruzi and other intracellular parasites are discussed.

Antichagasic Activity of Komaroviquinone Is Due to Generation of Reactive Oxygen Species Catalyzed by Trypanosoma cruzi Old Yellow Enzyme

ABSTRACT A novel potent trypanocidal diterpene, komaroviquinone, was reduced by Trypanosoma cruzi old yellow enzyme (TcOYE) to its semiquinone radical. The reductase activity in trypanosome lysates was completely immunoabsorbed by anti-TcOYE antibody. Since TcOYE is expressed throughout the T. cruzi life cycle, komaroviquinone is an interesting candidate for developing new antichagasic drugs.

CARBAMOYL-PHOSPHATE SYNTHETASE II IN KINETOPLASTIDS

Genes for carbamoyl-phosphate synthetase II (CPS II), the first enzyme of de novo pyrimidine biosynthesis, were cloned from kinetoplastids, Trypanosoma cruzi and Leishmania mexicana. T. cruzi CPS II gene encodes a protein of 1524 amino acids that encompasses the glutaminase and CPS domains, but incorporates neither aspartate carbamoyltransferase nor dihydroorotase. The residue corresponding to lysine 993 of Escherichia coli CPS, a residue that characterizes the CPS inhibited by UMP and that is replaced by tryptophan in those inhibited by UTP, is in kinetoplastids a hydrophilic glutamine, in line with the preferential inhibition by UDP of kinetoplastid CPS II.

Cloning and functional expression of Rpn1, a regulatory-particle non-ATPase subunit 1, of proteasome from Trypanosoma cruzi

Non-lysosomal protein degradation in eukaryotic cells involves a proteolytic complex referred to as 26S proteasome that consists of a 20S core particle and one or two 19S regulatory particles. We have cloned the gene RPN1 encoding Rpnl (regulatory-particle non-ATPase subunit 1), one of the largest subunits of proteasome, from Trypanosoma cruzi. It contains 2712 bp and encodes 904 amino acid residues with a calculated molecular mass of 98.2 kDa and an isoelectric point of 5.2. The predicted amino acid sequence of the trypanosomatid Rpn1 shares 39.0 and 32.0% overall identities with human Rpn1 and Saccharomyces cerevisiae Nas1 (non-ATPase subunit 1), an Rpn1 homolog, respectively, while the sequence identities among T. cruzi, Plasmodium falciparum, and Entamoeba histolytica Rpnl are approximately 30%. T. cruzi Rpn1 contains nine repeats of about 36 amino acid residues conserved in Rpn1s from various organisms. T. cruzi RPN1 is located on the 2300- and 1900-kb chromosomal DNA, displays a putative allelic variation as RPN1-1 and RPN1-2 with 98.8% identity between these two putative gene products, and is transcribed from both alleles at a comparable level throughout the three developmental stages of the parasite, epimastigotes, trypomastigotes, and amastigotes. The expression of the trypanosomatid Rpnl in the temperature-sensitive nas1 yeast mutant rescued the growth defect at the restrictive temperature, indicating that Rpn1 functions as a Nas1 and probably assembles into the 19S regulatory particle of the yeast 26S proteasome.

Quantitative determination of Trypanosoma cruzi growth inside host cells in vitro and effect of allopurinol.

Trypanosoma cruzi, the parasitic protozoan flagellate that causes Chagas1 disease against which a prominent chemotherapy is urgently needed, occurs as two different developmental stages in mammalian hosts (Brener, 1973; de Souza, 1984). The nondividing and infective trypomastigote form, which possesses a flagellum for locomotion, circulates in the bloodstream and the amastigote form, which has no free flagellum, proliferates in the cytoplasm of host cells, resulting in the destruction of infected cells. To identify the basis of a rational chemotherapy, it is of importance to analyze the biological and biochemical differences between these parasitic forms and host cells, since little is known about the molecular and cellular mechanisms involved in their morphological changes and the interactions that exist between the parasites and host cells. For this purpose, we have constructed an in vitro culture system of host cells infected with T. cruzi for the quantitative determination of time course of parasite growth. This system has enabled us to test allopurinol and other agents for their efficacies in inhibiting the parasite growth inside the host cells.

Identification of alkylbenzene sulfonate surfactants leaching from an acrylonitrile butadiene rubber as novel inhibitors of calcineurin activity.

Calcineurin (CN) is a Ca(2+)/calmodulin (CaM) dependent serine/threonine protein phosphatase and plays important role in several cellular functions in both higher and lower eukaryotes. Here we report inhibition of CN by linear alkylbenzene sulfonate. The clue to the finding was obtained while identifying the inhibitory material leaching from acrylonitrile butadiene rubber used for packing. Using standard dodecylbenzene sulfonate (C12-LAS), we obtained strong inhibition of CN with a half maximal inhibitory concentration of 9.3 µM, whereas analogs such as p-octylbenzene sulfonate and SDS hardly or only slightly affected CN activity. Three alkaline phosphatases, derived from shrimp, bacteria, and calf-intestine, which exhibit similar enzymatic activities to CN, were not inhibited by C12-LAS at concentrations of up to 100 µM. Furthermore, C12-LAS did not inhibit Ca(2+)/CaM-dependent myosin light chain kinase activity when tested at concentrations of up to 36 µM. The results indicate that C12-LAS is a potent selective inhibitor of CN activity.

Derivatives of Dictyostelium discoideum differentiation-inducing factor-3 suppress the activities of Trypanosoma cruzi in vitro and in vivo.

Chagas disease (human American trypanosomiasis), which is caused by the protozoan parasite Trypanosoma cruzi, is responsible for numerous deaths each year; however, established treatments for the disease are limited. Differentiation-inducing factor-1 (DIF-1) and DIF-3 are chlorinated alkylphenones originally found in the cellular slime mold Dictyostelium discoideum that have been shown to possess pharmacological activities. Here, we investigated the effects of DIF-3 derivatives on the infection rate and growth of T. cruzi by using an in vitro assay system utilizing host human fibrosarcoma HT1080 cells. Certain DIF-3 derivatives, such as butoxy-DIF-3 (Bu-DIF-3), at micro-molar levels strongly suppressed both the infection rate and growth of T. cruzi in HT1080 cells and exhibited little toxicity for HT1080 cells. For example, the IC50 of DIF-3 and Bu-DIF-3 versus the growth of T. cruzi in HT1080 cells were 3.95 and 0.72μM, respectively, and the LD50 of the two compounds versus HT1080 cells were both greater than 100μM. We also examined the effects of DIF-3 and Bu-DIF-3 on T. cruzi activity in C57BL/6 mice. Intraperitoneally administered Bu-DIF-3 (50mg/kg) significantly suppressed the number of trypomastigotes in blood with no apparent adverse effects. These results strongly suggest that DIF-3 derivatives could be new lead compounds in the development of anti-trypanosomiasis drugs.

Effects of synthetic undecapeptides on Trypanosoma cruzi in vitro

Abstract KLKLLLLLKLK-NH 2 is a potent antibacterial peptide based on a core peptide in sapecin B isolated from Salcophaga peregrina . The activities of the undecapeptide l -KLKLLLLLKLK-NH 2 and its d -enantiomer against Trypanosoma cruzi were evaluated in an in vitro culture system using mammalian host cells (HeLa) infected with the parasites in terms of their effects on intracellular replication by the parasite and the host-cell infection rate. The rate of infection of HeLa cells by T. cruzi on day 2 after infection was 54.7%, which decreased to 39.8 and 10.7% by the addition of 2 and 20 μg ml −1 l -KLKLLLLLKLK-NH 2 , respectively. The d -enantiomer had a similar inhibitory effect. The addition of undecapeptides, especially the l -enantiomer, to trypomastigotes brought about a markedly lowered viability, suggesting a direct killing effect on trypomastigotes, probably different from the mechanism by which purine analogs inhibit amastigote proliferation. l -KLKLLLLLKLK-NH 2 produced a dose-dependent increase in cytosolic free calcium concentration ([Ca 2+ ] i ) in trypomastigotes that was not inhibited by Ca 2+ channel blockers and was dependent on extracellular Ca 2+ . These results suggest that the peptide has a greater affinity for the plasma membrane of trypomastigotes than for that of host cells, producing elevated membrane permeability that decreases the rate of infection by T. cruzi .

Inhibition by 3′-Azido-3′-Deoxythymidine (AZT) of trypanosoma Cruzi Growth in Mammalian Cells and a Possible Mechanism of Action

In mammalian hosts, the protozoan parasite Trypanosoma cruzi, which causes Chagas’ disease in Latin America, occurs in two different forms, trypomastigotes and amastigotes. Trypomastigotes circulate in the bloodstream, invading host cells and transforming into amastigotes. Amastigotes grow by binary fission, and eventually transform back to trypomastigotes that destroy the host cells and again appear in the circulation. We have recently established an in vitro culture system of HeLa cells infected with T. cruzi, that enables us to determine quantitatively the time course of parasite growth inside the host cells (N.-Shimada et al., 1994 & 1996). This in vitro system is now available as a primary screening for anti-T. cruzi compounds. We expect that effective compounds may decrease the infection rate of host cells and/or the number of amastigotes per infected HeLa. Here we report the inhibitory action of an anti-human immunodeficiency virus (anti-HIV) agent, AZT, on the growth of T. cruzi in mammalian cells in vitro

Inhibition of autolysosome formation in host autophagy by Trypanosoma cruzi infection

Autophagy has emerged as an essential component of the defense system against intracellular pathogens. We demonstrated that Trypanosoma cruzi, an intracellular protozoan parasite, was not eliminated by the hosts autophagic machinery despite exposure to the host cell cytoplasm. Puncta of microtubule-associated protein 1 light chain 3 (LC3), an autophagy marker, and LC3-II, a lipidated form of LC3, were significantly increased after infection with T. cruzi, indicating that the parasite activated the early steps of host autophagy and induced autophagosome formation. However, autolysosomes were not observed in the infected cells. In addition, T. cruzi was not enwrapped by autophagosomes, suggesting that the parasite has mechanisms to allow it to evade autophagic capture. The results of this study indicate that host autophagy is incomplete following T. cruzi infection.