RESUMO
African trypanosomes (Trypanosoma brucei) have a digenetic lifecycle that alternates between the mammalian bloodstream and the tsetse fly vector. In the bloodstream, replicating long slender parasites transform into non-dividing short stumpy forms. Upon transmission into the fly midgut, short stumpy cells differentiate into actively dividing procyclics. A hallmark of this process is the replacement of the bloodstream-stage surface coat composed of variant surface glycoprotein (VSG) with a new coat composed of procyclin. Pre-existing VSG is shed by a zinc metalloprotease activity (MSP-B) and glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC). We now provide a detailed analysis of the coordinate and inverse regulation of these activities during synchronous differentiation. MSP-B mRNA and protein levels are upregulated during differentiation at the same time as proteolysis whereas GPI-PLC levels decrease. When transcription or translation is inhibited, VSG release is incomplete and a substantial amount of protein stays cell-associated. Both modes of release are still evident under these conditions, but GPI hydrolysis plays a quantitatively minor role during normal differentiation. Nevertheless, GPI biosynthesis shifts early in differentiation from a GPI-PLC sensitive structure to a resistant procyclic-type anchor. Translation inhibition also results in a marked increase in the mRNA levels of both MSP-B and GPI-PLC, consistent with negative regulation by labile protein factors. The relegation of short stumpy surface GPI-PLC to a secondary role in differentiation suggests that it may play a more important role as a virulence factor within the mammalian host.
Assuntos
Regulação da Expressão Gênica , Glicoproteínas de Membrana/metabolismo , Metaloproteases/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/crescimento & desenvolvimento , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo , Animais , Glicosilfosfatidilinositol Diacilglicerol-Liase , Estágios do Ciclo de Vida , Glicoproteínas de Membrana/genética , Metaloproteases/genética , Camundongos , Fosfatidilinositol Diacilglicerol-Liase , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/citologia , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/genéticaRESUMO
IPC (inositol phosphorylceramide) synthase is an enzyme essential for fungal viability, and it is the target of potent antifungal compounds such as rustmicin and aureobasidin A. Similar to fungi and some other lower eukaryotes, the protozoan parasite Trypanosoma cruzi is capable of synthesizing free or protein-linked glycoinositolphospholipids containing IPC. As a first step towards understanding the importance and mechanism of IPC synthesis in T. cruzi, we investigated the effects of rustmicin and aureobasidin A on the proliferation of different life-cycle stages of the parasite. The compounds did not interfere with the axenic growth of epimastigotes, but aureobasidin A decreased the release of trypomastigotes from infected murine peritoneal macrophages and the number of intracellular amastigotes in a dose-dependent manner. We have demonstrated for the first time that all forms of T. cruzi express an IPC synthase activity that is capable of transferring inositol phosphate from phosphatidylinositol to the C-1 hydroxy group of C6-NBD-cer {6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-amino]hexanoylceramide} to form inositol phosphoryl-C6-NBD-cer, which was purified and characterized by its chromatographic behaviour on TLC and HPLC, sensitivity to phosphatidylinositol-specific phospholipase C and resistance to mild alkaline hydrolysis. Unlike the Saccharomyces cerevisiae IPC synthase, the T. cruzi enzyme is stimulated by Triton X-100 but not by bivalent cations, CHAPS or fatty-acid-free BSA, and it is not inhibited by rustmicin or aureobasidin A, or the two in combination. Further studies showed that aureobasidin A has effects on macrophages independent of the infecting T. cruzi cells. These results suggest that T. cruzi synthesizes its own IPC, but by a mechanism that is not affected by rustmicin and aureobasidin A.
Assuntos
Hexosiltransferases/antagonistas & inibidores , Hexosiltransferases/metabolismo , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologia , Animais , Antifúngicos/farmacologia , Células Cultivadas , Depsipeptídeos/farmacologia , Fungos/efeitos dos fármacos , Fungos/enzimologia , Técnicas In Vitro , Lactonas/farmacologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Macrófagos/microbiologia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Microssomos/enzimologia , Proteínas de Protozoários/metabolismo , Ratos , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
In this study, we characterized ceramide synthase (CerS) of the protozoan parasite Trypanosoma cruzi at the molecular and functional levels. TcCerS activity was detected initially in a cell-free system using the microsomal fraction of epimastigote forms of T. cruzi, [(3)H]dihydrosphingosine or [(3)H]sphingosine, and fatty acids or acyl-CoA derivatives as acceptor or donor substrates, respectively. TcCerS utilizes both sphingoid long-chain bases, and its activity is exclusively dependent on acyl-CoAs, with palmitoyl-CoA being preferred. In addition, Fumonisin B(1), a broad and well-known acyl-CoA-dependent CerS inhibitor, blocked the parasite's CerS activity. However, unlike observations in fungi, the CerS inhibitors Australifungin and Fumonisin B(1) did not affect the proliferation of epimastigotes in culture, even after exposure to high concentrations or after extended periods of treatment. A search of the parasite genome with the conserved Lag1 motif from Lag1p, the yeast acyl-CoA-dependent CerS, identified a T. cruzi candidate gene (TcCERS1) that putatively encodes the parasite's CerS activity. The TcCERS1 gene was able to functionally complement the lethality of a lag1Δ lac1Δ double deletion yeast mutant in which the acyl-CoA-dependent CerS is not detectable. The complemented strain was capable of synthesizing normal inositol-containing sphingolipids and is 10 times more sensitive to Fumonisin B(1) than the parental strain.
Assuntos
Genoma de Protozoário , Oxirredutases/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/enzimologia , Acil Coenzima A/metabolismo , Acil Coenzima A/farmacologia , Clonagem Molecular , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/genética , Meios de Cultura , Ativação Enzimática , Ensaios Enzimáticos , Fumonisinas/farmacologia , Genes de Protozoários , Teste de Complementação Genética , Oxirredutases/antagonistas & inibidores , Oxirredutases/genética , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Filogenia , Proteínas de Protozoários/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Deleção de Sequência , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Esfingosina/farmacologia , Tetra-Hidronaftalenos/farmacologia , Fatores de Tempo , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/genéticaRESUMO
This paper describes the design, synthesis and pharmacological evaluation of new N-acylhydrazone (NAH) compounds, belonging to the N-substituted-phenyl-1,2,3-triazole-4-acylhydrazone class (2a-p). Classical heteroaromatic ring bioisosterism strategies were applied to the previously reported N-phenylpyrazolyl-4-acylhydrazone derivative 1, elected as lead-compound due to its important anti-aggregating profile on arachidonic acid induced platelet aggregation (IC(50)=24+/-0.5 micro M), from which emerge this new series 2. These new compounds 2a-p were readily synthesized, characterized and tested on platelet aggregation assays induced by collagen (5 micro g/mL), ADP (5 micro M) and arachidonic acid (100 micro M) in rabbit citrated platelet-rich plasma. Compounds 2b, 2d, and 2h were found to be the most potent, exhibiting a significant antiplatelet activity on arachidonic acid- and collagen-induced platelet aggregation. In addition, these new antiplatelet agents are free of gastric ulcerogenic effect and presented discrete anti-inflammatory and analgesic properties. The N-para-chlorophenyltriazolyl-4-acylhydrazone compound 2h produced the highest inhibitory effect on collagen (IC(50)=21.6+/-0.4 micro M) and arachidonic acid-induced platelet aggregation (IC(50)=2.2+/-0.06 micro M), suggesting that the nature of the substituent on the phenyl ring of the N-heteroaromatic system of NAH moiety may be an important structural requirement for the improvement of antiplatelet activity, in comparison with lead-series 1.