RESUMO
Human defensins play a fundamental role in the initiation of innate immune responses to some microbial pathogens. Here we show that colonic epithelial model HCT116 cells respond to Trypanosoma cruzi infection by secreting defensin α-1, which reduces infection. We also report the early effects of defensin α-1 on invasive trypomastigotes that involve damage of the flagellar structure to inhibit parasite motility and reduce cellular infection. Short exposure of defensin α-1 to trypomastigotes shows that defensin α-1 binds to the flagellum, resulting in flagellar membrane and axoneme alterations, followed by breaking of the flagellar membrane connected to the trypanosome body, leading to detachment and release of the parasite flagellum. In addition, defensin α-1 induces a significant reduction in parasite motility in a peptide concentration-dependent manner, which is abrogated by anti-defensin α-1 IgG. Preincubation of trypomastigotes with a concentration of defensin α-1 that inhibits 50% trypanosome motility significantly reduced cellular infection by 80%. Thus, human defensin α-1 is an innate immune molecule that is secreted by HCT116 cells in response to T. cruzi infection, inhibits T. cruzi motility, and plays an important role in reducing cellular infection. This is the first report showing a novel cellular innate immune response to a human parasite by secretion of defensin α-1, which neutralizes the motility of a human parasite to reduce cellular infection. The mode of activity of human defensin α-1 against T. cruzi and its function may provide insights for the development of new antiparasitic strategies.
Assuntos
Células Epiteliais/imunologia , Células Epiteliais/parasitologia , Flagelos/imunologia , Locomoção , Trypanosoma cruzi/imunologia , alfa-Defensinas/metabolismo , Membrana Celular/ultraestrutura , Flagelos/fisiologia , Flagelos/ultraestrutura , Células HCT116 , Humanos , Trypanosoma cruzi/fisiologia , Trypanosoma cruzi/ultraestruturaRESUMO
Trypanosoma cruzi causes Chagas disease (American trypanosomiasis), which threatens the lives of millions of people and remains incurable in its chronic stage. The antifungal drug posaconazole that blocks sterol biosynthesis in the parasite is the only compound entering clinical trials for the chronic form of this infection. Crystal structures of the drug target enzyme, Trypanosoma cruzi sterol 14alpha-demethylase (CYP51), complexed with posaconazole, another antifungal agent fluconazole and an experimental inhibitor, (R)-4'-chloro-N-(1-(2,4-dichlorophenyl)-2-(1H-imid-azol-1-yl)ethyl)biphenyl-4-carboxamide (VNF), allow prediction of important chemical features that enhance the drug potencies. Combined with comparative analysis of inhibitor binding parameters, influence on the catalytic activity of the trypanosomal enzyme and its human counterpart, and their cellular effects at different stages of the Trypanosoma cruzi life cycle, the structural data provide a molecular background to CYP51 inhibition and azole resistance and enlighten the path for directed design of new, more potent and selective drugs to develop an efficient treatment for Chagas disease.
Assuntos
Antifúngicos/química , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/química , Triazóis/química , Trypanosoma cruzi/enzimologia , Animais , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Doença de Chagas/tratamento farmacológico , Cristalografia por Raios X , Humanos , Immunoblotting , Estrutura Molecular , Miócitos Cardíacos/parasitologia , Estrutura Secundária de Proteína , Triazóis/farmacologia , Triazóis/uso terapêutico , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/patogenicidadeRESUMO
Trypanosoma cruzi, the causative agent of Chagas' disease, infects heart and muscle cells leading to cardiac arrest, followed by death. The genetic architectures in the early T. cruzi infection process of human cells are unknown. To understand the genetic architectures of the early invasion process of T. cruzi, we conducted gene transcription microarray analysis, followed by gene network construction of the host cell response in primary human coronary artery smooth muscle (HCASM) cells infected with T. cruzi or exposed to T. cruzi gp83, a ligand used by the trypanosome to bind host cells. Using seven RT-PCR verified up-regulated genes (FOSB, ATF5, INPP1, CCND2, THBS1, LAMC1, and APLP2) as the seed for network construction, we built an interaction network of the early T. cruzi infection process containing 165 genes, connected by 598 biological interactions. This interactome network is centered on the BCL6 gene as a hub. Silencing the expression of two seed genes (THBS1 and LAMC1) by RNAi reduced T. cruzi infection. Overall, our results elucidate the significant and complex process involved in T. cruzi infection of HCASM cells at the transcriptome level. This is the first elucidation into the interactome network in human cells caused by T. cruzi and its gp83 ligand.
Assuntos
Vasos Coronários/parasitologia , Redes Reguladoras de Genes , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/parasitologia , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/fisiologia , Vasos Coronários/citologia , Perfilação da Expressão Gênica , Humanos , Ligantes , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNA , Transcrição Gênica , Regulação para CimaRESUMO
Trypanosoma cruzi, the causative agent of Chagas heart disease, infects heart and other cells leading to cardiac arrest frequently followed by death. The disease affects millions of individuals in the Americas and is posing health problems because of blood transmission in the US due to large Latin American immigration. Since the current drugs present serious side effects and do not cure the chronic infection, it is critically important to understand the early process of cellular infection at the molecular and structural levels to design novel inhibitors to block T. cruzi infection. In this review, the authors critically analyze the molecular and cellular basis of early T. cruzi infection and discuss the future directions in this area. The candidate T. cruzi invasive genes and host genes involved in the process of early infection are just beginning to be understood. The trypanosome invasive proteins are excellent targets for intervention. The progress made in the cell biology of T. cruzi infection will also facilitate the development of novel cell-based therapies to ameliorate the disease.
Assuntos
Trypanosoma cruzi/genética , Animais , Caseína Quinase II/metabolismo , Doença de Chagas/enzimologia , Doença de Chagas/genética , Cisteína Endopeptidases/metabolismo , Glicoproteínas/genética , Interações Hospedeiro-Parasita , Humanos , Neuraminidase/genética , Prolil Oligopeptidases , Proteínas de Protozoários , Serina Endopeptidases/metabolismo , Trypanosoma cruzi/enzimologiaRESUMO
Sterol 14alpha-demethylases (CYP51) serve as primary targets for antifungal drugs, and specific inhibition of CYP51s in protozoan parasites Trypanosoma brucei (TB) and Trypanosoma cruzi (TC) might provide an effective treatment strategy for human trypanosomiases. Primary inhibitor selection is based initially on the cytochrome P450 spectral response to ligand binding. Ligands that demonstrate strongest binding parameters were examined as inhibitors of reconstituted TB and TC CYP51 activity in vitro. Direct correlation between potency of the compounds as CYP51 inhibitors and their antiparasitic effect in TB and TC cells implies essential requirements for endogenous sterol production in both trypanosomes and suggests a lead structure with a defined region most promising for further modifications. The approach developed here can be used for further large-scale search for new CYP51 inhibitors.
Assuntos
Antiprotozoários/farmacologia , Inibidores das Enzimas do Citocromo P-450 , Oxirredutases/antagonistas & inibidores , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Tripanossomíase/tratamento farmacológico , Animais , Antiprotozoários/uso terapêutico , Esterol 14-Desmetilase , Especificidade por Substrato , Trypanosoma brucei brucei/citologia , Trypanosoma brucei brucei/enzimologia , Trypanosoma cruzi/citologia , Trypanosoma cruzi/enzimologiaRESUMO
The cytochrome P540 (CYP) superfamily currently includes about 9000 proteins forming more than 800 families. The enzymes catalyze monooxygenation of a vast array of compounds and play essentially two roles. They provide biodefense (detoxification of xenobiotics, antibiotic production) and participate in biosynthesis of important endogenous molecules, particularly steroids. Based on these two roles, sterol 14/*alpha*/-demethylases (CYP51) belong to the second group of P450s. The CYP51 family, however, is very special as its members preserve strict functional conservation in enzyme activity in all biological kingdoms. At amino acid identity across the kingdoms as low as 25-30%, they all catalyze essentially the same three-step reaction of oxidative removal of the 14/*alpha*/-methyl group from the lanostane frame. This reaction is the required step in sterol biosynthesis of pathogenic microbes. We have shown that specific inhibition of protozoan CYP51 can potentially provide treatment for human trypanosomiases. Three sets of CYP51 inhibitors tested in vitro and in trypanosomal cells in this study include azoles [best results being 50% cell growth inhibition at <1 and at 1.3 muM for Trypanosoma cruzi (TC) and Trypanosoma brucei (TB), respectively], non-azole compounds (50% TC cell growth inhibition at 5 microM) and substrate analogs of the 14/*alpha*/-demethylase reaction. 32-Methylene cyclopropyl lanost-7-enol exhibited selectivity toward TC with 50% cell growth inhibition at 3 microM.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma/enzimologia , Animais , Azóis/química , Azóis/metabolismo , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/genética , Desenho de Fármacos , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Esteróis/química , Esteróis/metabolismo , Especificidade por Substrato , Trypanosoma/citologia , Trypanosoma/fisiologiaRESUMO
Interactions between Trypanosoma cruzi and the extracellular matrix play an important role in cellular invasion. Here we show that T. cruzi increases the levels of thrombospondin-1 (TSP-1) expression in host cells during early infection. Stable RNA interference of host cell TSP-1 knocks down the levels of TSP-1 transcripts and protein expression in mammalian cells causing inhibition of T. cruzi infection. Addition of TSP-1 to these cells restores infection. Thus, host TSP-1, regulated by the parasite, plays a crucial role in early infection. This is the first report showing that a human parasite modulates TSP-1 expression to facilitate infection.
Assuntos
Doença de Chagas/genética , Interferência de RNA , Trombospondina 1/genética , Trypanosoma cruzi , Animais , Células Cultivadas , Doença de Chagas/metabolismo , Humanos , Trombospondina 1/biossíntese , Trombospondina 1/farmacologiaRESUMO
It has been shown that the invasive trypomastigote forms of Trypanosoma cruzi use and modulate components of the extracellular matrix (ECM) during the initial process of infection. Infective trypomastigotes up-regulate the expression of laminin γ-1 (LAMC1) and thrombospondin (THBS1) to facilitate the recruitment of trypomastigotes to enhance cellular infection. Silencing the expression of LAMC1 and THBS1 by stable RNAi dramatically reduces trypanosome infection. T. cruzi gp83, a ligand that mediates the attachment of trypanosomes to cells to initiate infection, up-regulates LAMC1 expression to enhance cellular infection. Infective trypomastigotes interact with LAMC1 through galectin-3 (LGALS3), a human lectin, to enhance cellular infection. Silencing the expression of LGALS3 also reduces cellular infection. Some trypanosome surface molecules also interact with the ECM to facilitate infection. Despite the role of the ECM in T. cruzi infection, almost nothing is known about the ECM interactome networks operating in the process of T. cruzi infection. In this mini review, we critically analyze and discuss the regulation of the ECM by T. cruzi and its gp83 ligand, and present the first elucidation of the human ECM interactome network, regulated by T. cruzi and its gp83 ligand, to facilitate cellular infection. The elucidation of the human ECM interactome regulated by T. cruzi is critically important to the understanding of the molecular pathogenesis of T. cruzi infection and developing novel approaches of intervention in Chagas' disease.
RESUMO
Trypanosoma cruzi (TC) causes Chagas disease, which in its chronic stage remains incurable. We have shown recently that specific inhibition of TC sterol 14alpha-demethylase (TCCYP51) with imidazole derivatives is effective in killing both extracellular and intracellular human stages of TC. An alternative set of TCCYP51 inhibitors has been identified using optical high throughput screening followed by web-database search for similar structures. The best TCCYP51 inhibitor from this search was found to have structural similarity to a class of cyclooxygenase-2-selective inhibitors, the indomethacin-amides. A number of indomethacin-amides were found to bind to TCCYP51, inhibit its activity in vitro, and produce strong antiparasitic effects in the cultured TC cells. Analysis of TC sterol composition indicated that the mode of action of the compounds is by inhibition of sterol biosynthesis in the parasite.
Assuntos
Amidas/química , Amidas/farmacologia , Inibidores das Enzimas do Citocromo P-450 , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Indometacina/análogos & derivados , Trypanosoma cruzi/enzimologia , Animais , Antiparasitários/química , Antiparasitários/farmacologia , Avaliação Pré-Clínica de Medicamentos , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/enzimologia , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/enzimologia , Ligantes , Esterol 14-Desmetilase , Esteróis/química , Esteróis/metabolismo , Trypanosoma cruzi/citologia , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Nuclear factor erythroid 2-related factor 2 (Nrf2) mediates the transcriptional response of cells to oxidative stress and is translocated into the nucleus following, or concomitant with, its activation by electrophiles or reactive oxygen species. The mechanism of its translocation into the nucleus is not entirely elucidated. Here we have identified two novel nuclear localization signal (NLS) motifs in murine Nrf2, one located near the N-terminal region (amino acid residues 42-53) and the other (residues 587-593) located near the C-terminal region. Imaging of green fluorescent protein (GFP)-tagged Nrf2 revealed that mutation(s) in any of these sequences resulted in decreased nuclear fluorescence intensity compared with the wild-type Nrf2 when Nrf2 activation was induced with the electrophile tert-butylhydroquinone. The mutations also impaired Nrf2-induced transactivation of antioxidant response element-driven reporter gene expression to the same extent as the Nrf2 construct bearing mutation in a previously identified bipartite NLS that maps at residues 494-511. When linked to GFP or to GFP-PEPCK-C each of the novel NLS motifs was sufficient to drive nuclear translocation of the fusion proteins. Co-immunoprecipitation assays demonstrated that importins alpha5 and beta1 associate with Nrf2, an interaction that was blocked by the nuclear import inhibitor SN50. SN50 also blocked tert-butylhydroquinone-induced nuclear fluorescence of GFP-Nrf2 in cells transfected with wild-type GFP-Nrf2. Overall these results reveal that multiple NLS motifs in Nrf2 function in its nuclear translocation in response to pro-oxidant stimuli and that the importin alpha-beta heterodimer nuclear import receptor system plays a critical role in the import process.
Assuntos
Núcleo Celular/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Sinais de Localização Nuclear/metabolismo , Estresse Oxidativo/fisiologia , Elementos de Resposta/fisiologia , Ativação Transcricional/fisiologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Motivos de Aminoácidos/fisiologia , Núcleo Celular/genética , Inibidores Enzimáticos/farmacologia , Humanos , Hidroquinonas/farmacologia , Células K562 , Mutação , Fator 2 Relacionado a NF-E2/genética , Sinais de Localização Nuclear/genética , Peptídeos/farmacologia , Ativação Transcricional/efeitos dos fármacos , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMO
Human defensins play a fundamental role in the initiation of innate immune responses to some microbial pathogens. Here we show that human defensin alpha-1 displays a trypanocidal role against Trypanosoma cruzi, the causative agent of Chagas' disease. The toxicity of human defensin alpha-1 against T. cruzi is mediated by membrane pore formation and the induction of nuclear and mitochondrial DNA fragmentation, leading to trypanosome destruction. Exposure of trypomastigote and amastigote forms of T. cruzi to defensin alpha-1 significantly reduced parasite viability in a peptide concentration-dependent and saturable manner. The toxicity of defensin alpha-1 against T. cruzi is blocked by anti-defensin alpha-1 immunoglobulin G. Electron microscopic analysis of trypomastigotes exposed to defensin alpha-1 revealed pore formation in the cellular and flagellar membranes, membrane disorganization, and blebbing as well as cytoplasmic vacuolization. Furthermore, human defensin alpha-1 enters the trypanosome when membrane pores are present and is associated with later intracellular damage. Trypanosome membrane depolarization abolished the toxicity of defensin alpha-1 against the parasite. Preincubation of trypomastigotes with defensin alpha-1 followed by exposure to human epithelial cells significantly reduced T. cruzi infection in these cells. Thus, human defensin alpha-1 is an innate immune molecule that causes severe toxicity to T. cruzi and plays an important role in reducing cellular infection. This is the first report showing that human defensin alpha-1 causes membrane pore formation in a human parasite, leading to trypanosome destruction.
Assuntos
Fragmentação do DNA , Trypanosoma cruzi/fisiologia , alfa-Defensinas/toxicidade , Animais , Membrana Celular/ultraestrutura , Citoplasma/ultraestrutura , Células Epiteliais/parasitologia , Células HeLa , Humanos , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Trypanosoma cruzi/imunologia , Trypanosoma cruzi/patogenicidade , Trypanosoma cruzi/ultraestrutura , Vacúolos/ultraestrutura , alfa-Defensinas/imunologiaRESUMO
It is thought that Trypanosoma cruzi, the protozoan that causes Chagas' disease, modulates the extracellular matrix network to facilitate infection of human cells. However, direct evidence to document this phenomenon is lacking. Here we show that the T. cruzi gp83 ligand, a cell surface trans-sialidase-like molecule that the parasite uses to attach to host cells, increases the level of laminin gamma-1 transcript and its expression in mammalian cells, leading to an increase in cellular infection. Stable RNA interference (RNAi) with host cell laminin gamma-1 knocks down the levels of laminin gamma-1 transcript and protein expression in mammalian cells, causing a dramatic reduction in cellular infection by T. cruzi. Thus, host laminin gamma-1, which is regulated by the parasite, plays a crucial role in the early process of infection. This is the first report showing that knocking down the expression of a human gene by RNAi inhibits the infection of an intracellular parasite.
Assuntos
Inativação Gênica/efeitos dos fármacos , Laminina/antagonistas & inibidores , Proteínas de Protozoários/genética , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/metabolismo , Doença de Chagas/prevenção & controle , Humanos , Laminina/genética , Laminina/metabolismo , Neuraminidase/química , Neuraminidase/genética , Proteínas de Protozoários/química , RNA Interferente Pequeno/uso terapêutico , Trypanosoma cruzi/genéticaRESUMO
In this work, we report the cloning and characterization of the first cell surface casein kinase II (CKII) substrate (Tc-1) of Trypanosoma cruzi, the causative agent of Chagas' disease. Analysis of the gene sequence revealed a 1,653-bp open reading frame coding for 550 amino acid residues. Northern blot analysis showed a 4.5-kb transcript that is expressed in invasive trypomastigotes but not in noninvasive epimastigote forms of T. cruzi. Southern blot analysis indicates that Tc-1 is a single-copy gene. At the amino acid level, Tc-1 displayed 95% and 99% identity to two hypothetical proteins recently reported by the T. cruzi genome project. Analysis of the translated amino acid sequence indicates that the Tc-1 gene has a putative transmembrane domain with multiple cytoplasmic and extracellular CKII phosphosites. Exogenous human CKII was able to phosphorylate serine residues on both recombinant Tc-1 and Tc-1 of intact trypomastigotes. This phosphorylation was inhibited by the CKII inhibitors heparin and 4,5,6,7,-tetrabromo-2-azabenzimidazole. Immunoblots of solubilized trypomastigotes, epimastigotes, and amastigotes probed with anti-recombinant Tc-1 immunoglobulin G revealed a 62-kDa protein that is expressed only in infective trypomastigotes. Immunoprecipitation of labeled surface proteins of trypomastigotes indicated that the 62-kDa protein is a surface protein, and we found that the protein is uniformly distributed on the surface of trypomastigotes by direct immunofluorescence. Antibodies to Tc-1 effectively blocked trypomastigote invasion of host cells and consequently reduced parasite load. Preincubation of either trypomastigotes or myoblasts with CKII inhibitors blocked T. cruzi infection. Thus, for the first time, we describe a cell surface CKII substrate of a protozoan parasite that is phosphorylated by human CKII and that is involved in cellular infection.
Assuntos
Caseína Quinase II/metabolismo , Clonagem Molecular , Proteínas de Membrana/genética , Mioblastos Cardíacos/parasitologia , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Sequência de Aminoácidos , Animais , Caseína Quinase II/química , Caseína Quinase II/genética , Caseína Quinase II/isolamento & purificação , Humanos , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Peso Molecular , Mioblastos Cardíacos/enzimologia , Fosforilação , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Ratos , Especificidade por Substrato , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/patogenicidade , Fatores de Virulência/genética , Fatores de Virulência/isolamento & purificaçãoRESUMO
Human galectin-3 binds to the surface of Trypanosoma cruzi trypomastigotes and human coronary artery smooth muscle (CASM) cells. CASM cells express galectin-3 on their surface and secrete it. Exogenous galectin-3 increased the binding of T. cruzi to CASM cells. Trypanosome binding to CASM cells was enhanced when either T. cruzi or CASM cells were preincubated with galectin-3. Cells stably transfected with galectin-3 antisense show a dramatic decrease in galectin-3 expression and very little T. cruzi adhesion to cells. The addition of galectin-3 to these cells restores their initial capacity to bind to trypanosomes. Thus, host galectin-3 expression is required for T. cruzi adhesion to human cells and exogenous galectin-3 enhances this process, leading to parasite entry.