RESUMO
Trypanosoma cruzi invades non-professional phagocytic cells by subverting their membrane repair process, which is dependent on membrane injury and cell signaling, intracellular calcium increase, and lysosome recruitment. Cells lacking lysosome-associated membrane proteins 1 and 2 (LAMP1 and LAMP2) are less permissive to parasite invasion but more prone to parasite intracellular multiplication. Several passages through a different intracellular environment can significantly change T. cruzi's gene expression profile. Here, we evaluated whether one single passage through LAMP-deficient (KO) or wild-type (WT) fibroblasts, thus different intracellular environments, could influence T. cruzi Y strain trypomastigotes' ability to invade L6 myoblasts and WT fibroblasts host cells. Parasites released from LAMP2 KO cells (TcY-L2-/-) showed higher invasion, calcium signaling, and membrane injury rates, for the assays in L6 myoblasts, when compared to those released from WT (TcY-WT) or LAMP1/2 KO cells (TcY-L1/2-/-). On the other hand, TcY-L1/2-/- showed higher invasion, calcium signaling, and cell membrane injury rates, for the assays in WT fibroblasts, compared to TcY-WT and TcY-L1/2-/-. Albeit TcY-WT presented an intermediary invasion and calcium signaling rates, compared to the others, in WT fibroblasts, they induced lower levels of injury, which reinforces that signals mediated by surface membrane protein interactions also have a significant contribution to trigger host cell calcium signals. These results clearly show that parasites released from WT or LAMP KO cells are distinct from each other. Additionally, these parasites' ability to invade the cell may be distinct depending on which cell type they interact with. Since these alterations most likely would reflect differences among parasite surface molecules, we also evaluated their proteome. We identified few protein complexes, membrane, and secreted proteins regulated in our dataset. Among those are some members of MASP, mucins, trans-sialidases, and gp63 proteins family, which are known to play an important role during parasite infection and could correlate to TcY-WT, TcY-L1/2-/-, and TcY-L2-/- biological behavior.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Animais , Células Cultivadas , Doença de Chagas/patologia , Fibroblastos/parasitologia , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteínas de Membrana Lisossomal/genética , Lisossomos , Proteínas de Membrana , Camundongos , Mioblastos/parasitologiaRESUMO
Infection by Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, depends on reactive oxygen species (ROS), which has been described to induce parasite proliferation in mammalian host cells. It is unknown how the parasite manages to increase host ROS levels. Here, we found that intracellular T. cruzi forms release in the host cytosol its major cyclophilin of 19 kDa (TcCyp19). Parasites depleted of TcCyp19 by using CRISPR/Cas9 gene replacement proliferate inefficiently and fail to increase ROS, compared to wild type parasites or parasites with restored TcCyp19 gene expression. Expression of TcCyp19 in L6 rat myoblast increased ROS levels and restored the proliferation of TcCyp19 depleted parasites. These events could also be inhibited by cyclosporin A, (a cyclophilin inhibitor), and by polyethylene glycol-linked to antioxidant enzymes. TcCyp19 was found more concentrated in the membrane leading edges of the host cells in regions that also accumulate phosphorylated p47phox , as observed to the endogenous cyclophilin A, suggesting some mechanisms involved with the translocation process of the regulatory subunit p47phox in the activation of the NADPH oxidase enzymatic complex. We concluded that cyclophilin released in the host cell cytosol by T. cruzi mediates the increase of ROS, required to boost parasite proliferation in mammalian hosts.
Assuntos
Ciclofilinas/metabolismo , Citosol/metabolismo , Interações Hospedeiro-Parasita , Espécies Reativas de Oxigênio/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismo , Animais , Ciclofilinas/biossíntese , Ciclofilinas/genética , Citosol/química , Mioblastos/parasitologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Ratos , Trypanosoma cruzi/genéticaRESUMO
Trypanosoma cruzi P21 protein (P21) is a putative secreted and immunomodulatory molecule with potent bioactive properties such as induction of phagocytosis and actin cytoskeleton polymerization. Despite the bioactive properties described so far, the action of P21 on parasite replication in muscle cell lineage or T. cruzi parasitism during acute experimental infection is unclear. We observed that recombinant P21 (rP21) decreased the multiplication of T. cruzi in C2C12 myoblasts, phenomenon associated with greater actin polymerization and IFN-γ and IL-4 higher expression. During experimental infection, lower cardiac nests, inflammatory infiltrate and fibrosis were observed in mice infected and treated with rP21. These results were correlated with large expression of IFN-γ counterbalanced by high levels of IL-10, which was consistent with the lower cardiac tissue injury found in these mice. We have also observed that upon stress, such as that induced by the presence of the IFN-γ cytokine, T. cruzi produced more P21. The effect of P21 in controlling the replication of T. cruzi, may indicate an evolutionary mechanism of survival developed by the parasite. Thus, when subjected to different stress conditions, the protozoan produces more P21, which induces T. cruzi latency in the host organism, enabling the protozoan to evade the host's immune system.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Malária/parasitologia , Mioblastos/parasitologia , Miocárdio/patologia , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/fisiologia , Doença Aguda , Animais , Linhagem Celular , Interações Hospedeiro-Parasita , Humanos , Evasão da Resposta Imune , Peptídeos e Proteínas de Sinalização Intercelular/genética , Interferon gama/metabolismo , Malária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Carga Parasitária , Proteínas de Protozoários/genéticaRESUMO
The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease. This neglected tropical disease causes severe morbidity and mortality in endemic regions. About 30% of T. cruzi infected individuals will present with cardiac complications. Invasive trypomastigotes released from infected cells can be carried in the vascular endothelial system to infect neighboring and distant cells. During the process of cellular infection, the parasite induces host cells, to increase the levels of host thrombospondin-1 (TSP-1), to facilitate the process of infection. TSP-1 plays important roles in the functioning of vascular cells, including vascular endothelial cells with important implications in cardiovascular health. Many signal transduction pathways, including the yes-associated protein 1 (YAP)/transcriptional coactivator, with PDZ-binding motif (TAZ) signaling, which are upstream of TSP-1, have been linked to the pathophysiology of heart damage. The molecular mechanisms by which T. cruzi signals, and eventually infects, heart endothelial cells remain unknown. To evaluate the importance of TSP-1 expression in heart endothelial cells during the process of T. cruzi infection, we exposed heart endothelial cells prepared from Wild Type and TSP-1 Knockout mouse to invasive T. cruzi trypomastigotes at multiple time points, and evaluated changes in the hippo signaling cascade using immunoblotting and immunofluorescence assays. We found that the parasite turned off the hippo signaling pathway in TSP-1KO heart endothelial cells. The levels of SAV1 and MOB1A increased to a maximum of 2.70 ± 0.23 and 5.74 ± 1.45-fold at 3 and 6 h, respectively, in TSP-1KO mouse heart endothelial cells (MHEC), compared to WT MHEC, following a parasite challenge. This was accompanied by a significant continuous increase in the nuclear translocation of downstream effector molecule YAP, to a maximum mean nuclear fluorescence intensity of 10.14 ± 0.40 at 6 h, compared to wild type cells. Furthermore, we found that increased nuclear translocated YAP significantly colocalized with the transcription co-activator molecule pan-TEAD, with a maximum Pearson's correlation coefficient of 0.51 ± 0.06 at 6 h, compared to YAP-Pan-TEAD colocalization in the WT MHEC, which decreased significantly, with a minimum Pearson's correlation coefficient of 0.30 ± 0.01 at 6 h. Our data indicate that, during the early phase of infection, upregulated TSP-1 is essential for the regulation of the hippo signaling pathway. These studies advance our understanding of the molecular interactions occurring between heart endothelial cells and T. cruzi, in the presence and absence of TSP-1, providing insights into processes linked to parasite dissemination and pathogenesis.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Células Endoteliais/parasitologia , Mioblastos/parasitologia , Miocárdio/citologia , Proteínas de Protozoários/fisiologia , Trombospondina 1/fisiologia , Trypanosoma cruzi/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais/metabolismo , Técnicas de Inativação de Genes , Camundongos , Mioblastos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Transdução de Sinais/fisiologia , Trombospondina 1/deficiência , Transativadores/fisiologiaRESUMO
Trypanosoma protists are pathogens leading to a spectrum of devastating infectious diseases. The range of available chemotherapeutics against Trypanosoma is limited, and the existing therapies are partially ineffective and cause serious adverse effects. Formation of the PEX14-PEX5 complex is essential for protein import into the parasites' glycosomes. This transport is critical for parasite metabolism and failure leads to mislocalization of glycosomal enzymes, with fatal consequences for the parasite. Hence, inhibiting the PEX14-PEX5 protein-protein interaction (PPI) is an attractive way to affect multiple metabolic pathways. Herein, we have used structure-guided computational screening and optimization to develop the first line of compounds that inhibit PEX14-PEX5 PPI. The optimization was driven by several X-ray structures, NMR binding data, and molecular dynamics simulations. Importantly, the developed compounds show significant cellular activity against Trypanosoma, including the human pathogen Trypanosoma brucei gambiense and Trypanosoma cruzi parasites.
Assuntos
Proteínas de Membrana/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Piridinas/síntese química , Piridinas/farmacologia , Tripanossomicidas/síntese química , Tripanossomicidas/farmacologia , Animais , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/biossíntese , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mioblastos/efeitos dos fármacos , Mioblastos/parasitologia , Proteínas de Protozoários/biossíntese , Ratos , Relação Estrutura-Atividade , Trypanosoma brucei gambiense/efeitos dos fármacos , Trypanosoma brucei gambiense/metabolismo , Trypanosoma brucei rhodesiense/efeitos dos fármacosRESUMO
INTRODUCTION: Chagas disease, the most important parasitic infection in Latin America, is caused by the intracellular protozoan Trypanosoma cruzi. To treat this disease, only two nitroheterocyclic compounds with toxic side effects exist and frequent treatment failures are reported. Hence there is an urgent need to develop new drugs. Recently, metabolomics has become an efficient and cost-effective strategy for dissecting drug mode of action, which has been applied to bacteria as well as parasites, such as different Trypanosome species and forms. OBJECTIVES: We assessed if the metabolomics approach can be applied to study drug action of the intracellular amastigote form of T. cruzi in a parasite-host cell system. METHODS: We applied a metabolic fingerprinting approach (DI-MS and NMR) to evaluate metabolic changes induced by six different (candidate) drugs in a parasite-host cell system. In a second part of our study, we analyzed the impact of two drugs on polar metabolites, lipid and proteins to evaluate if affected pathways can be identified. RESULTS: Metabolic signatures, obtained by the fingerprinting approach, resulted in three different clusters. Two can be explained by already known of mode actions, whereas the three experimental drugs formed a separate cluster. Significant changes induced by drug action were observed in all the three metabolic fractions (polar metabolites, lipids and proteins). We identified a general impact on the TCA cycle, but no specific pathways could be attributed to drug action, which might be caused by a high percentage of common metabolome between a eukaryotic host cell and a eukaryotic parasite. Additionally, ion suppression effects due to differences in abundance between host cells and parasites may have occurred. CONCLUSION: We validated the metabolic fingerprinting approach to a complex host-cell parasite system. This technique can potentially be applied in the early stage of drug discovery and could help to prioritize early leads or reconfirmed hits for further development.
Assuntos
Interações Hospedeiro-Parasita , Metabolômica/métodos , Mioblastos/parasitologia , Proteômica/métodos , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Linhagem Celular , Metabolismo dos Lipídeos , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Metaboloma , Mioblastos/metabolismo , Proteoma/química , Ratos , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/patogenicidadeRESUMO
Some 1,3-diarylureas and 1-((1,4-trans)-4-aryloxycyclohexyl)-3-arylureas (cHAUs) activate heme-regulated kinase causing protein synthesis inhibition via phosphorylation of the eukaryotic translation initiation factor 2 (eIF2) in mammalian cancer cells. To evaluate if these agents have potential to inhibit trypanosome multiplication by also affecting the phosphorylation of eIF2 alpha subunit (eIF2α), we tested 25 analogs of 1,3-diarylureas and cHAUs against Trypanosoma cruzi, the agent of Chagas disease. One of them (I-17) presented selectivity close to 10-fold against the insect replicative forms and also inhibited the multiplication of T. cruzi inside mammalian cells with an EC50 of 1-3 µM and a selectivity of 17-fold. I-17 also prevented replication of African trypanosomes (Trypanosoma brucei bloodstream and procyclic forms) at similar doses. It caused changes in the T. cruzi morphology, arrested parasite cell cycle in G1 phase, and promoted phosphorylation of eIF2α with a robust decrease in ribosome association with mRNA. The activity against T. brucei also implicates eIF2α phosphorylation, as replacement of WT-eIF2α with a non-phosphorylatable eIF2α, or knocking down eIF2 protein kinase-3 by RNAi increased resistance to I-17. Therefore, we demonstrate that eIF2α phosphorylation can be engaged to develop trypanosome-static agents in general, and particularly by interfering with activity of eIF2 kinases.
Assuntos
Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/metabolismo , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/metabolismo , Ureia/metabolismo , Ureia/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Doença de Chagas/microbiologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Fase G1/efeitos dos fármacos , Heme/metabolismo , Humanos , Mioblastos/efeitos dos fármacos , Mioblastos/parasitologia , Testes de Sensibilidade Parasitária , Fosforilação , Ratos , Ureia/análogos & derivados , eIF-2 Quinase/metabolismoRESUMO
Nematophin, a known antibiotic natural product against Staphylococcus aureus for almost 20 years, is produced by all strains of Xenorhabdus nematophila. Despite its simple structure, its biosynthesis was unknown. Its biosynthetic pathway is reported using heterologous production in Escherichia coli. Additionally, the identification, structure elucidation, and biosynthesis of six extended nematophin derivatives from Xenorhabdus PB62.4 carrying an additional valine are reported. Preliminary bioactivity studies suggest a biological role of these compounds in the bacteria-nematode-insect symbiosis.
Assuntos
Antibacterianos/biossíntese , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Antiparasitários/química , Antiparasitários/metabolismo , Antiparasitários/farmacologia , Escherichia coli/metabolismo , Indóis/química , Indóis/metabolismo , Indóis/farmacologia , Leishmania/efeitos dos fármacos , Mioblastos/efeitos dos fármacos , Mioblastos/parasitologia , Nematoides/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Ratos , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade , Trypanosoma/efeitos dos fármacos , Xenorhabdus/metabolismoRESUMO
The malaria-causing parasite Plasmodium falciparum employs a salvage pathway for the biosynthesis of nucleotides, in contrast to de novo biosynthesis that is utilized by the human host. A series of twenty-two 2-, 6- and 5'-modified adenosine ribonucleosides was synthesized, with the expectation that these compounds would generate toxic metabolites instead of active nucleotides by the pathogen, while remaining inert in host cells. Bioassays with P. falciparum (K1 strain) indicated IC50 values as low as 110nM and a selectivity index with respect to cytotoxicity toward an L6 rat myoblast cell line of >1000 for the most potent analogue.
Assuntos
Antimaláricos/farmacologia , Malária/tratamento farmacológico , Mioblastos/efeitos dos fármacos , Nucleosídeos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Animais , Antimaláricos/síntese química , Antimaláricos/química , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Mioblastos/parasitologia , Nucleosídeos/síntese química , Nucleosídeos/química , Testes de Sensibilidade Parasitária , Ratos , Relação Estrutura-AtividadeRESUMO
The contractile vacuole complex (CVC) of Trypanosoma cruzi, the etiologic agent of Chagas disease, collects and expels excess water as a mechanism of regulatory volume decrease after hyposmotic stress; it also has a role in cell shrinking after hyperosmotic stress. Here, we report that, in addition to its role in osmoregulation, the CVC of T. cruzi has a role in the biogenesis of acidocalcisomes. Expression of dominant-negative mutants of the CVC-located small GTPase Rab32 (TcCLB.506289.80) results in lower numbers of less-electron-dense acidocalcisomes, lower content of polyphosphate, lower capacity for acidocalcisome acidification and Ca(2+) uptake that is driven by the vacuolar proton pyrophosphatase and the Ca(2+)-ATPase, respectively, as well as less-infective parasites, revealing the role of this organelle in parasite infectivity. By using fluorescence, electron microscopy and electron tomography analyses, we provide further evidence of the active contact of acidocalcisomes with the CVC, indicating an active exchange of proteins between the two organelles.
Assuntos
Ácidos/metabolismo , Cálcio/metabolismo , Doença de Chagas/parasitologia , Organelas/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/patogenicidade , Animais , Western Blotting , Células Cultivadas , Chlorocebus aethiops , Fibroblastos/citologia , Fibroblastos/parasitologia , Imunofluorescência , Prepúcio do Pênis/citologia , Humanos , Técnicas Imunoenzimáticas , Masculino , Microscopia Eletrônica , Mioblastos/citologia , Mioblastos/parasitologia , Osmorregulação/fisiologia , Proteínas de Protozoários/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vacúolos/metabolismo , Células Vero , Equilíbrio HidroeletrolíticoRESUMO
BACKGROUND: Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, represents a very important public health problem in Latin America where it is endemic. Although mostly asymptomatic at its initial stage, after the disease becomes chronic, about a third of the infected patients progress to a potentially fatal outcome due to severe damage of heart and gut tissues. There is an urgent need for new drugs against Chagas disease since there are only two drugs available, benznidazole and nifurtimox, and both show toxic side effects and variable efficacy against the chronic stage of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Genetically engineered parasitic strains are used for high throughput screening (HTS) of large chemical collections in the search for new anti-parasitic compounds. These assays, although successful, are limited to reporter transgenic parasites and do not cover the wide T. cruzi genetic background. With the aim to contribute to the early drug discovery process against Chagas disease we have developed an automated image-based 384-well plate HTS assay for T. cruzi amastigote replication in a rat myoblast host cell line. An image analysis script was designed to inform on three outputs: total number of host cells, ratio of T. cruzi amastigotes per cell and percentage of infected cells, which respectively provides one host cell toxicity and two T. cruzi toxicity readouts. The assay was statistically robust (Z´ values >0.6) and was validated against a series of known anti-trypanosomatid drugs. CONCLUSIONS/SIGNIFICANCE: We have established a highly reproducible, high content HTS assay for screening of chemical compounds against T. cruzi infection of myoblasts that is amenable for use with any T. cruzi strain capable of in vitro infection. Our visual assay informs on both anti-parasitic and host cell toxicity readouts in a single experiment, allowing the direct identification of compounds selectively targeted to the parasite.
Assuntos
Mioblastos/parasitologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Automação Laboratorial , Linhagem Celular , Chlorocebus aethiops , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Nifurtimox/farmacologia , Nitroimidazóis/farmacologia , RatosRESUMO
Chagas disease is a chronic infection caused by the protozoan parasite Trypanosoma cruzi, manifested in progressive cardiomyopathy and/or gastrointestinal dysfunction. Therapeutic options to prevent or treat Chagas disease are limited. CYP51, the enzyme key to the biosynthesis of eukaryotic membrane sterols, is a validated drug target in both fungi and T. cruzi. Sulfonamide derivatives of 4-aminopyridyl-based inhibitors of T. cruzi CYP51 (TcCYP51), including the sub-nanomolar compound 3, have molecular structures distinct from other validated CYP51 inhibitors. They augment the biologically relevant chemical space of molecules targeting TcCYP51. In a 2.08 Å X-ray structure, TcCYP51 is in a conformation that has been influenced by compound 3 and is distinct from the previously characterized ground-state conformation of CYP51 drug-target complexes. That the binding site was modulated in response to an incoming inhibitor for the first time characterizes TcCYP51 as a flexible target rather than a rigid template.
Assuntos
Inibidores de 14-alfa Desmetilase/farmacologia , Esterol 14-Desmetilase/metabolismo , Sulfonamidas/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Inibidores de 14-alfa Desmetilase/síntese química , Inibidores de 14-alfa Desmetilase/química , Animais , Relação Dose-Resposta a Droga , Camundongos , Modelos Moleculares , Estrutura Molecular , Mioblastos/efeitos dos fármacos , Mioblastos/parasitologia , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química , Tripanossomicidas/síntese química , Tripanossomicidas/química , Trypanosoma cruzi/enzimologiaRESUMO
BACKGROUND: Trypanosoma cruzi is the etiological agent of Chagas disease, a debilitating illness that affects millions of people in the Americas. A major finding of the T. cruzi genome project was the discovery of a novel multigene family composed of approximately 1,300 genes that encode mucin-associated surface proteins (MASPs). The high level of polymorphism of the MASP family associated with its localization at the surface of infective forms of the parasite suggests that MASP participates in host-parasite interactions. We speculate that the large repertoire of MASP sequences may contribute to the ability of T. cruzi to infect several host cell types and/or participate in host immune evasion mechanisms. METHODS: By sequencing seven cDNA libraries, we analyzed the MASP expression profile in trypomastigotes derived from distinct host cells and after sequential passages in acutely infected mice. Additionally, to investigate the MASP antigenic profile, we performed B-cell epitope prediction on MASP proteins and designed a MASP-specific peptide array with 110 putative epitopes, which was screened with sera from acutely infected mice. FINDINGS AND CONCLUSIONS: We observed differential expression of a few MASP genes between trypomastigotes derived from epithelial and myoblast cell lines. The more pronounced MASP expression changes were observed between bloodstream and tissue-culture trypomastigotes and between bloodstream forms from sequential passages in acutely infected mice. Moreover, we demonstrated that different MASP members were expressed during the acute T. cruzi infection and constitute parasite antigens that are recognized by IgG and IgM antibodies. We also found that distinct MASP peptides could trigger different antibody responses and that the antibody level against a given peptide may vary after sequential passages in mice. We speculate that changes in the large repertoire of MASP antigenic peptides during an infection may contribute to the evasion of host immune responses during the acute phase of Chagas disease.
Assuntos
Doença de Chagas/parasitologia , Regulação da Expressão Gênica , Interações Hospedeiro-Parasita , Proteínas de Protozoários/biossíntese , Trypanosoma cruzi/genética , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/imunologia , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/parasitologia , Perfilação da Expressão Gênica , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Proteínas de Membrana/biossíntese , Proteínas de Membrana/imunologia , Camundongos , Mioblastos/parasitologia , Proteínas de Protozoários/imunologiaRESUMO
BACKGROUND: Toxoplasma gondii belongs to a large and diverse group of obligate intracellular parasitic protozoa. Primary culture of mice skeletal muscle cells (SkMC) was employed as a model for experimental toxoplasmosis studies. The myogenesis of SkMC was reproduced in vitro and the ability of T. gondii tachyzoite forms to infect myoblasts and myotubes and its influence on SkMC myogenesis were analyzed. RESULTS: In this study we show that, after 24 h of interaction, myoblasts (61%) were more infected with T. gondii than myotubes (38%) and inhibition of myogenesis was about 75%. The role of adhesion molecules such as cadherin in this event was investigated. First, we demonstrate that cadherin localization was restricted to the contact areas between myocytes/myocytes and myocytes/myotubes during the myogenesis process. Immunofluorescence and immunoblotting analysis of parasite-host cell interaction showed a 54% reduction in cadherin expression at 24 h of infection. Concomitantly, a reduction in M-cadherin mRNA levels was observed after 3 and 24 h of T. gondii-host cell interaction. CONCLUSIONS: These data suggest that T. gondii is able to down regulate M-cadherin expression, leading to molecular modifications in the host cell surface that interfere with membrane fusion and consequently affect the myogenesis process.
Assuntos
Caderinas/antagonistas & inibidores , Desenvolvimento Muscular , Músculo Esquelético/patologia , Músculo Esquelético/parasitologia , Toxoplasma/patogenicidade , Animais , Células Cultivadas , Perfilação da Expressão Gênica , Interações Hospedeiro-Parasita , Camundongos , Fibras Musculares Esqueléticas/parasitologia , Fibras Musculares Esqueléticas/patologia , Mioblastos/parasitologia , Mioblastos/fisiologiaRESUMO
Phosphoinositide phospholipase C (PI-PLC) plays an essential role in cell signaling. A unique Trypanosoma cruzi PI-PLC (TcPI-PLC) is lipid modified in its N terminus and localizes to the outer surface of the plasma membrane of amastigotes. We show here that TcPI-PLC is developmentally regulated in amastigotes and shows two peaks of surface expression during the developmental cycle of T. cruzi, the first immediately after differentiation of trypomastigotes into amastigotes and the second before differentiation of amastigotes into trypomastigotes. Surface expression of TcPI-PLC coincides with phosphatidylinositol 4,5-bisphosphate (PIP(2)) depletion in the host cell membrane and with an increase in the levels of its product, inositol 1,4,5-trisphosphate. During extracellular differentiation, PI-PLC is secreted into the incubation medium. Maximal early expression of TcPI-PLC on the surface of amastigotes and PIP(2) depletion coincide with host cytoskeletal changes, Ca(2+) signaling, and transcriptional responses described previously. The presence of TcPI-PLC on the outer surface of the plasma membrane of the parasite and the capacity to be secreted and to alter host phospholipids are novel mechanisms of the host-parasite interaction.
Assuntos
Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfoinositídeo Fosfolipase C/metabolismo , Trypanosoma cruzi/enzimologia , Animais , Linhagem Celular , Membrana Celular/enzimologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Interações Hospedeiro-Parasita , Hidrólise , Fosfatos de Inositol/metabolismo , Mioblastos/metabolismo , Mioblastos/parasitologia , Fosfoinositídeo Fosfolipase C/genética , Transdução de SinaisRESUMO
We examined the extent to which different Trypanosoma cruzi strains induce transcriptomic changes in cultured L(6)E(9) myoblasts 72 hours after infection with Brazil (TC I), Y (TC II), CL (TC II), and Tulahuen (TC II) strains. Expression of 6,289 distinct, fully annotated unigenes was quantified with 27,000 rat oligonucleotide arrays in each of the four replicas of all control and infected RNA samples. Considering changes greater than 1.5-fold and P values < 0.05, the Tulahuen strain was the most disruptive to host transcriptome (17% significantly altered genes), whereas the Y strain altered only 6% of the genes. The significantly altered genes in the infected cells were largely different among the strains, and only 21 genes were similarly changed by all four strains. However, myoblasts infected with different strains showed proportional overall gene-expression alterations. These results indicate that infection with different parasite strains modulates similar but not identical pathways in the host cells.
Assuntos
Perfilação da Expressão Gênica , Mioblastos/metabolismo , Mioblastos/parasitologia , Transcrição Gênica , Trypanosoma cruzi , Animais , Linhagem Celular , Humanos , Ratos , Trypanosoma cruzi/classificação , Trypanosoma cruzi/genéticaRESUMO
American trypanosomiasis (Chagas disease) continues to be a significant public health problem, and the therapeutic potential of current antichagasic agents (nifurtimox and benznidazole) is rather limited. Here we report on the antitrypanosomal effect of 1-methoxyspirobrassinol and other indole phytoalexins--secondary metabolites produced by Cruciferous plants. These compounds, that previously demonstrated antimicrobial and anticancer properties, displayed significant antiproliferative effects on intracellular amastigotes of Trypanosoma cruzi and may be prospective candidates for antichagasic drug design and development.
Assuntos
Antiprotozoários/farmacologia , Indóis/farmacologia , Terpenos/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Avaliação Pré-Clínica de Medicamentos , Indóis/química , Concentração Inibidora 50 , Mioblastos/efeitos dos fármacos , Mioblastos/parasitologia , Ratos , Sesquiterpenos , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Relação Estrutura-Atividade , Tiazóis/química , Tiazóis/farmacologia , Tiocarbamatos/química , Tiocarbamatos/farmacologia , FitoalexinasRESUMO
Infection with the parasite Trypanosoma cruzi causes Chagas disease. In this study we demonstrated that there was an increase in cyclin D1 expression in T. cruzi (Tulahuen strain)-infected myoblasts. To examine a possible mechanism for the increased cyclin D1 expression we transfected L(6)E(9) myoblasts with cyclin D1 luciferase reporter constructs and infected with T. cruzi. There was no evidence of an increase in promoter activity. Additionally, quantitative PCR did not demonstrate any change in cyclin D1 message during infection. Moreover, we demonstrated that the cyclin D1 protein was significantly stabilized after infection. Collectively, these data indicate that infection with T. cruzi increases cyclin D1 protein abundance post-translationally.
Assuntos
Doença de Chagas/metabolismo , Ciclina D1/metabolismo , Regulação da Expressão Gênica , Mioblastos/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Ciclina D1/genética , Primers do DNA/genética , Humanos , Luciferases , Mioblastos/parasitologiaRESUMO
Analogues of the natural antibiotic thiolactomycin (TLM), an inhibitor of the condensing reactions of type II fatty acid synthase, were synthesized and evaluated for their ability to inhibit the growth of the malaria parasite Plasmodium falciparum. Alkylation of the C4 hydroxyl group led to the most significant increase in growth inhibition (over a 100-fold increase in activity compared to TLM). To investigate the mode of action, the P. falciparum KASIII enzyme was produced for inhibitor assay. A number of TLM derivatives were identified that showed improved inhibition of this enzyme compared to TLM. Structure-activity relationships for enzyme inhibition were identified for some series of TLM analogues, and these also showed weak correlation with inhibition of parasite growth, but this did not hold for other series. On the basis of the lack of a clear correlation between inhibition of pfKASIII activity and parasite growth, we conclude that pfKASIII is not the primary target of TLM analogues. Some of the analogues also inhibited the growth of the parasitic protozoa Trypanosoma cruzi, T. brucei, and Leishmania donovani.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/antagonistas & inibidores , Antimaláricos/síntese química , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/química , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , Acetiltransferases/antagonistas & inibidores , Acetiltransferases/química , Acetiltransferases/genética , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Linhagem Celular , Ácido Graxo Sintase Tipo II , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/genética , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Mioblastos/efeitos dos fármacos , Mioblastos/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Ratos , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/química , Tiofenos/farmacologia , Tripanossomicidas/síntese química , Tripanossomicidas/química , Tripanossomicidas/farmacologiaRESUMO
Although Chagas' disease is known to provoke severe acute myositis, information on muscle regeneration is missing. The current paper shows that during T. cruzi infection in rats, skeletal muscle parasitism and the consequent inflammatory process are higher in muscle with a high proportion of type-I myofibres (soleus and diaphragm). Immunohistochemistry showed an acute inflammatory process characterized by ED1+ and ED2+ macrophages, CD8+ lymphocytes, and NK cells. Parasite-nest rupture provoked segmental degeneration of myofibres followed by regeneration. These phenomena were observed at both light and transmission electron microscopy levels. Myofibre regeneration involved activation of satellite cells assessed by the expression of MyoD, a muscle-specific transcription factor. Ultrastructural evidence of fusion of myoblast-like cells with the intact segment of degenerating fibres has been provided. At the chronic phase no signs of fibrosis were found, but sparse and small inflammatory foci were found. Our results argue against the relevant participation of autoimmunity phenomena in both acute and chronic phases and furnish a new view for explaining histopathological findings in human patient muscles.