RESUMO
The protozoan parasite Leishmania mexicana causes chronic cutaneous disease in humans and most mouse strains. We previously showed that STAT4-deficient mice, but not IL-12p40-deficient mice, have more parasites and progressively growing lesions unlike those of wild-type mice, the lesions and parasite burdens of which plateau by 10-12 weeks post-infection. This demonstrates a STAT4-dependent, IL-12/IL-23-independent pathway of parasite control. Type I IFNs are important in viral and other infections and can activate STAT4. We found that IFN-alpha/betaR-deficient mice have a nonpersistent, early IFN-gamma defect, and a persistent, early IL-10 defect, without changes in serum IL-12 or LN-derived nitric oxide. We found less IL-10 per cell in CD25+CD4+ T cells and possibly fewer IL-10-producing cells in the draining LN of IFN-alpha/betaR-deficient vs. wild-type mice. IFN-alpha/betaR-deficient mice have chronic, nonprogressive disease, like wild-type mice, suggesting that IL-10 and IFN-gamma defects may balance each other. Our data indicate that although type I IFNs help promote early Th1 responses, they are not the missing activators of STAT4 responsible for partial control of L. mexicana. Also, the lack of lesion resolution in IFN-alpha/betaR-deficient mice despite lower IL-10 levels indicates that other pathways independent of T cell IL-10 help prevent an IL-12-driven clearance of parasites.
Assuntos
Interferon Tipo I/imunologia , Interferon gama/biossíntese , Interleucina-10/biossíntese , Leishmania mexicana/imunologia , Leishmaniose Cutânea/imunologia , Animais , Linfócitos T CD4-Positivos/química , Linfócitos T CD4-Positivos/imunologia , Interleucina-12/sangue , Subunidade alfa de Receptor de Interleucina-2/análise , Linfonodos/imunologia , Camundongos , Camundongos Knockout , Receptor de Interferon alfa e beta/deficiênciaRESUMO
Leishmania amazonensis induces a nonhealing infection in C3H mice, whereas infection with Leishmania major is self-healing. We found that C3H mice infected with L. amazonensis exhibited decreased IL-12 production, which could account for the susceptibility to this organism. However, exogenous IL-12 administration failed to induce a healing immune response. The failure of L. amazonensis-infected C3H mice to respond to IL-12 was associated with a specific defect in IL-12 receptor beta2 (IL-12Rbeta2) mRNA expression by CD4+ T cells. Furthermore, decreased IL-12Rbeta2 mRNA expression correlated with a decrease in the IL-12-signaling capacity of the lymph node (LN) cells. IL-4 did not contribute to susceptibility or down-regulation of the IL-12Rbeta2 subunit, because IL-4-/- mice remained susceptible to L. amazonensis infection, even after IL-12 administration, and CD4+ cells from infected IL-4-/- mice also had reduced expression of IL-12Rbeta2 mRNA. These results demonstrate that regulation of the IL-12 receptor, independent of IL-4, is a point of control for the immune response to leishmaniasis. In contrast to experimental L. major infections, where host genetics control susceptibility, these studies demonstrate that the lack of IL-12 responsiveness may be dictated by the pathogen, rather than the host.
Assuntos
Interleucina-12/antagonistas & inibidores , Interleucina-12/biossíntese , Interleucina-4/fisiologia , Leishmania mexicana/imunologia , Leishmaniose Cutânea/imunologia , Animais , Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/parasitologia , Linfócitos T CD4-Positivos/patologia , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Feminino , Predisposição Genética para Doença , Injeções Subcutâneas , Interferon gama/biossíntese , Interleucina-12/administração & dosagem , Interleucina-12/fisiologia , Interleucina-4/deficiência , Interleucina-4/genética , Leishmania major/imunologia , Leishmaniose Cutânea/etiologia , Leishmaniose Cutânea/genética , Leishmaniose Cutânea/metabolismo , Linfonodos/imunologia , Linfonodos/parasitologia , Linfonodos/patologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/biossíntese , Receptores de Interleucina/antagonistas & inibidores , Receptores de Interleucina/biossíntese , Receptores de Interleucina/deficiência , Receptores de Interleucina/genética , Receptores de Interleucina-12 , Transdução de Sinais/imunologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/metabolismoRESUMO
Glycosyl phosphatidylinositol (GPI) anchors in the bloodstream form of Trypanosoma brucei are unusual in that their two fatty acids are myristate. The myristates are added in the final stages of GPI biosynthesis in a remodeling reaction. Remodeling occurs first at the sn-2 position of glycerol, involving removal of a longer fatty acid and subsequent attachment of myristate. The second myristate is then incorporated into the sn-1 position, but the mechanism has been unclear due to the unavailability of a reliable cell-free system supporting complete remodeling. Here, we first refined the cell-free system (by removing Mn(2+) ions), thereby allowing efficient production of the dimyristoylated GPI precursor. Using this improved system, we made three new discoveries concerning the pathway for fatty acid remodeling. First, we discovered a monomyristoylated GPI (known as glycolipid theta') as an intermediate involved in remodeling at the sn-1 position. Second, we found an alternative pathway for production of glycolipid theta, the first lyso intermediate in remodeling. The alternative pathway involves an inositol-acylated GPI known as glycolipid lyso-C'. Finally, we found that there is significant breakdown of GPIs during remodeling in the cell-free system, and we speculate that this breakdown has a regulatory role in GPI biosynthesis.
Assuntos
Ácidos Graxos/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Ácido Mirístico/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Glicosilfosfatidilinositóis/biossíntese , CinéticaRESUMO
The survival of Trypanosoma cruzi, the causative agent of Chagas' disease, depends vitally on proteins and glycoconjugates that mediate the parasite/host interaction. Since most of these molecules are attached to the membrane by glycosylphosphatidylinositol (GPI), alternative means of chemotherapeutic intervention might emerge from GPI biosynthesis studies. The structure of the major 1G7 antigen GPI has been fully characterized by us (Güther, M. L. S., Cardoso de Almeida, M. L., Yoshida, N., and Ferguson, M. A. J.(1992) J. Biol. Chem. 267, 6820-6828; Heise, N., Cardoso de Almeida, M. L., and Ferguson, M. A. J.(1995) Mol. Biochem. Parasitol. 70, 71-84), and based on its properties we now report the complete precursor glycolipids predicted to be transferred to the nascent protein. Migrating closely to Trypanosoma brucei glycolipid A on TLC, such species, named glycolipids A-like 1 and A-like 2, were labeled with tritiated palmitic acid, myo-inositol, glucosamine, and mannose, but surprisingly only the less polar glycolipid A-like 1 incorporated ethanolamine. The predicted products following nitrous acid deamination and digestion with phospholipases A2, C, and D confirmed their GPI nature. Evidence that they may represent the anchor transferred to the 1G7 antigen came from the following analyses: (i) alpha-mannosidase treatments indicated that only one mannose was amenable to removal; (ii) their lipid moiety was identified as sn-1-alkyl-2-acylglycerol due to their sensitivity to phospholipase A2 (PLA2), mild base and by direct high performance TLC analysis of the corresponding benzoylated diradylglycerol components; and (iii) both glycolipids incorporated 3H-fatty acid only in the sn-2- and not in the sn-1-alkyl position as previously found in the GPI of the mature 1G7 antigen. Based on the differential [3H]ethanolamine incorporation pattern and the recent report that an aminoethylphosphonic acid (AEP) replaces ethanolamine phosphate (EtNH2-PO4) in the GPI in epimastigote sialoglycoproteins (Previato, J. O., Jones, C., Xavier, M. T., Wait, R., Travassos, L. R., Parodi, A. J., and Mendonça-Previato, L.(1995) J. Biol. Chem. 270, 7241-7250) it is proposed that glycolipid A-like 2 contains AEP and A-like 1 EtNH2-PO4. In the in vitro cell-free system both glycolipids were synthesized simultaneously and do not seem to bear a precursor/product relationship. Among the various components synthesized in vitro a glycolipid C-like corresponding to a form of glycolipid A-like 1 acylated on the inositol was also characterized. Phenylmethylsulfonyl fluoride, an inhibitor known to block the addition of ethanolamine phosphate in T. brucei but not in mammalian cells, also inhibits the synthesis of glycolipids A-like and C-like in T. cruzi, indicating that the putative trypanosome EtNH2-PO4/AEP transferase(s) might represent a potential target for chemotherapy.
Assuntos
Glicolipídeos/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Antígenos de Protozoários/metabolismo , Sequência de Carboidratos , Etanolamina , Etanolaminas/metabolismo , Glicerídeos/metabolismo , Glicosilfosfatidilinositóis/química , Cinética , Camundongos , Dados de Sequência Molecular , Ácido Palmítico , Ácidos Palmíticos/metabolismo , TrítioRESUMO
The glycosyl-phosphatidylinositol (GPI) anchor of the Trypanosoma brucei variant surface glycoprotein (VSG) is unique in having exclusively myristate as its fatty acid component. We previously demonstrated that the myristate specificity is the result of two independent pathways. First, the newly synthesized free GPI, which is not myristoylated, undergoes fatty acid remodeling to replace both its fatty acids with myristate. Second, the myristoylated precursor, glycolipid A, undergoes a myristate exchange reaction, detected by the replacement of unlabeled myristate by [3H]myristate. Remodeling and exchange have different enzymatic properties and apparently occur in different subcellular compartments. We now demonstrate that the GPI anchor linked to VSG is the major substrate for myristate exchange. VSG can be efficiently labeled with [3H]myristate by exchange in the presence of cycloheximide, an inhibitor that prevents new VSG synthesis and thus anchor addition to protein. Not only is newly synthesized VSG subject to exchange, but mature VSG, possibly recycling from the cell surface, also undergoes myristate exchange.
Assuntos
Glicosilfosfatidilinositóis/metabolismo , Ácidos Mirísticos/metabolismo , Processamento de Proteína Pós-Traducional , Trypanosoma brucei brucei/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo , Animais , Autorradiografia , Sistema Livre de Células , Cicloeximida/farmacologia , Eletroforese em Gel de Poliacrilamida , Cinética , Lipídeo A/metabolismo , Ácido Mirístico , Inibidores da Síntese de Proteínas/farmacologia , Trítio , Trypanosoma brucei brucei/efeitos dos fármacos , Glicoproteínas Variantes de Superfície de Trypanosoma/biossíntese , Glicoproteínas Variantes de Superfície de Trypanosoma/isolamento & purificaçãoRESUMO
The variant surface glycoprotein of African trypanosomes has a glycosyl phosphatidylinositol (GPI) anchor that is unusual in that its fatty acids are exclusively myristate. We showed previously that the myristate is added to a free GPI in a fatty acid remodeling reaction involving deacylation and reacylation, forming glycolipid A, the anchor precursor. We now demonstrate that trypanosomes have a second pathway for GPI anchor myristoylation distinct from the fatty acid remodeling pathway, which we call "myristate exchange." This reaction involves exchange of myristate into both the sn-1 and sn-2 positions of glycolipid A, which already contain myristate. Myristoyl-CoA, the probable myristate donor in the exchange reaction, has an apparent Km of about 6 nM. We have now identified a lyso-GPI, named theta', which has myristate as its sole fatty acid; the kinetics of formation and utilization of theta' are consistent with it being an intermediate in exchange. Myristate exchange and fatty acid remodeling appear to occur in different subcellular compartments, and the two reactions have different sensitivities to inhibitors. The myristate exchange reaction may be a proofreading system to ensure that the fatty acids on variant surface glycoproteins are exclusively myristate.
Assuntos
Glicosilfosfatidilinositóis/metabolismo , Ácidos Mirísticos/metabolismo , Trypanosoma/metabolismo , Acil Coenzima A/metabolismo , Animais , Membrana Celular/metabolismo , Sistema Livre de Células , Cromatografia em Camada Fina , Glicolipídeos/biossíntese , Glicolipídeos/isolamento & purificação , Glicosilfosfatidilinositóis/isolamento & purificação , Cinética , Ácido Mirístico , Fosfolipases ARESUMO
The variant surface glycoprotein (VSG) of T. brucei is anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor which is unique in that its fatty acids are exclusively myristate (a fourteen carbon saturated fatty acid). We showed that the myristate is added to the GPI precursor in a remodeling reaction involving deacylation and reacylation. We now demonstrate that trypanosomes have a second pathway of myristoylation for GPI anchors that we call "myristate exchange" which is distinct from the fatty acid remodeling pathway. We propose that this is an exchange of [3H]myristate into both sn-1 and sn-2 positions of glycolipid A, which already contains myristate, and have demonstrated this using inhibitors and a variety of other methods. We have partially characterized myristate exchange with respect to specificity and susceptibility to some inhibitors. The apparent Km for myristoyl CoA is 7 nM. This myristate-specific process may represent a proof-reading system to ensure that the fatty acids on VSG are exclusively myristate. Although myristate exchange was first discovered for glycolipid A, we now believe that VSG is the true substrate of this reaction. VSG is efficiently labeled by exchange in the presence of cycloheximide, which prevents anchoring of newly synthesized protein. Although its location is not yet known, we have evidence that exchange does not localize to either the endoplasmic reticulum or the plasma membrane. We will present data indicating that surface VSG may be internalized and undergo myristate exchange.
Assuntos
Glicosilfosfatidilinositóis/biossíntese , Miristatos/metabolismo , Trypanosoma brucei brucei/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo , Animais , Sistema Livre de Células , Trypanosoma brucei brucei/químicaRESUMO
The variant surface glycoprotein (VSG) of T. brucei is anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor which is unique in that its fatty acids are exclusively myristate (a fourteen carbon saturated fatty acid). We showed that the myristate is added to the GPI precursor in a remodeling reaction involving deacylation and reacylation. We now demonstrate that trypanosomes have a second pathway of myristoylation for GPI anchors that we call "myristate exchange" which is distinct from the fatty acid remodeling pathway. We propose that this is an exchange of [3H]myristate into both sn-1 and sn-2 positions of glycolipid A, which already contains myristate, and have demonstrated this using inhibitors and a variety of other methods. We have partially characterized myristate exchange with respect to specificity and susceptibility to some inhibitors. The apparent Km for myristoyl CoA is 7 nM. This myristate-specific process may represent a proof-reading system to ensure that the fatty acids on VSG are exclusively myristate. Although myristate exchange was first discovered for glycolipid A, we now believe that VSG is the true substrate of this reaction. VSG is efficiently labeled by exchange in the presence of cycloheximide, which prevents anchoring of newly synthesized protein. Although its location is not yet know, we have evidence that exchange does not localize to either the endoplasmic reticulum or the plasma membrane. We will present data indicating that surface VSG may be internalized and undergo myristate exchange
Assuntos
Animais , Fosfatidilinositóis/biossíntese , Glicolipídeos/biossíntese , Técnicas In Vitro , Miristatos/metabolismo , Trypanosoma brucei brucei/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/biossíntese , Acetatos/metabolismo , Ácidos Graxos/isolamento & purificação , Ácidos Graxos/metabolismo , Membrana Celular , Retículo Endoplasmático , CinéticaAssuntos
Glicosilfosfatidilinositóis/metabolismo , Trypanosoma brucei brucei/metabolismo , Acil Coenzima A/metabolismo , Animais , Sequência de Carboidratos , Glicosilfosfatidilinositóis/biossíntese , Glicosilfosfatidilinositóis/química , Dados de Sequência Molecular , Miristatos/metabolismo , Miristatos/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológico , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismoRESUMO
Trypanosoma brucei, the protozoan parasite responsible for African sleeping sickness, evades the host immune response through the process of antigenic variation. The variant antigen, known as the variant surface glycoprotein (VSG), is anchored to the cell surface by a glycosyl phosphatidylinositol (GPI) structure that contains myristate (n-tetradecanoate) as its only fatty acid component. The utilization of heteroatom-containing analogs of myristate was studied both in a cell-free system and in vivo. Results indicated that the specificity of fatty acid incorporation depends on chain length rather than on hydrophobicity. One analog, 10-(propoxy)decanoic acid, was highly toxic to trypanosomes in culture although it is nontoxic to mammalian cells.