RESUMO
Infection with obligatory intracellular bacteria is difficult to treat, as intracellular targets and delivery methods of therapeutics are not well known. Ehrlichia translocated factor-1 (Etf-1), a type IV secretion system (T4SS) effector, is a primary virulence factor for an obligatory intracellular bacterium, Ehrlichia chaffeensis In this study, we developed Etf-1-specific nanobodies (Nbs) by immunizing a llama to determine if intracellular Nbs block Etf-1 functions and Ehrlichia infection. Of 24 distinct anti-Etf-1 Nbs, NbD7 blocked mitochondrial localization of Etf-1-GFP in cotransfected cells. NbD7 and control Nb (NbD3) bound to different regions of Etf-1. Size-exclusion chromatography showed that the NbD7 and Etf-1 complex was more stable than the NbD3 and Etf-1 complex. Intracellular expression of NbD7 inhibited three activities of Etf-1 and E. chaffeensis: up-regulation of mitochondrial manganese superoxide dismutase, reduction of intracellular reactive oxygen species, and inhibition of cellular apoptosis. Consequently, intracellular NbD7 inhibited Ehrlichia infection, whereas NbD3 did not. To safely and effectively deliver Nbs into the host cell cytoplasm, NbD7 was conjugated to cyclized cell-permeable peptide 12 (CPP12-NbD7). CPP12-NbD7 effectively entered mammalian cells and abrogated the blockade of cellular apoptosis caused by E. chaffeensis and inhibited infection by E. chaffeensis in cell culture and in a severe combined-immunodeficiency mouse model. Our results demonstrate the development of an Nb that interferes with T4SS effector functions and intracellular pathogen infection, along with an intracellular delivery method for this Nb. This strategy should overcome current barriers to advance mechanistic research and develop therapies complementary or alternative to the current broad-spectrum antibiotic.
Assuntos
Ehrlichia chaffeensis/efeitos dos fármacos , Ehrlichiose/tratamento farmacológico , Anticorpos de Domínio Único/farmacologia , Sistemas de Secreção Tipo IV/genética , Animais , Apoptose/genética , Subpopulações de Linfócitos B/imunologia , Ehrlichia chaffeensis/genética , Ehrlichia chaffeensis/imunologia , Ehrlichia chaffeensis/patogenicidade , Ehrlichiose/genética , Ehrlichiose/imunologia , Ehrlichiose/patologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Anticorpos de Domínio Único/imunologia , Sistemas de Secreção Tipo IV/antagonistas & inibidores , Sistemas de Secreção Tipo IV/imunologia , Fatores de VirulênciaRESUMO
Ehrlichia chaffeensis is an obligatory intracellular bacterium that infects monocytes and macrophages, and causes human monocytic ehrlichiosis, an emerging life-threatening infectious disease. Ehrlichia translocated factor-1 (Etf-1), a type IV secretion system effector, is essential for Ehrlichia infection of host cells. Etf-1 translocates to mitochondria to block host apoptosis; furthermore, it can bind Beclin 1 (ATG6) to induce cellular autophagy and localize to E. chaffeensis-inclusion membrane to obtain host-cell cytoplasmic nutrients. In this study, we screened a synthetic library of over 320,000 cell-permeable macrocyclic peptides, which consist of an ensemble of random peptide sequences in the first ring and a small family of cell-penetrating peptides in the second ring, for Etf-1 binding. Library screening followed by hit optimization identified multiple Etf-1-binding peptides (with K D values of 1-10â µM) that efficiently enter the cytosol of mammalian cells. Peptides B7, C8, B7-131-5, B7-133-3, and B7-133-8 significantly inhibited Ehrlichia infection of THP-1 cells. Mechanistic studies revealed that peptide B7 and its derivatives inhibited the binding of Etf-1 to Beclin 1, and Etf-1 localization to E. chaffeensis-inclusion membranes, but not Etf-1 localization to the mitochondria. Our results not only affirm the critical role of Etf-1 functions in E. chaffeensis infection, but also demonstrate the feasibility of developing macrocyclic peptides as powerful chemical probes and potential treatment of diseases caused by Ehrlichia and other intracellular pathogens.
RESUMO
Upon oxidative stress, mammalian cells rapidly reprogram their translation. This is accompanied by the formation of stress granules (SGs), cytoplasmic ribonucleoprotein condensates containing untranslated mRNA molecules, RNA-binding proteins, 40S ribosomal subunits, and a set of translation initiation factors. Here we show that arsenite-induced stress causes a dramatic increase in the stop-codon readthrough rate and significantly elevates translation reinitiation levels on uORF-containing and bicistronic mRNAs. We also report the recruitment of translation termination factors eRF1 and eRF3, as well as ribosome recycling and translation reinitiation factors ABCE1, eIF2D, MCT-1, and DENR to SGs upon arsenite treatment. Localization of these factors to SGs may contribute to a rapid resumption of mRNA translation after stress relief and SG disassembly. It may also suggest the presence of post-termination, recycling, or reinitiation complexes in SGs. This new layer of translational control under stress conditions, relying on the altered spatial distribution of translation factors between cellular compartments, is discussed.
Assuntos
Arsenitos , Animais , Códon de Terminação , Arsenitos/farmacologia , Arsenitos/metabolismo , Ribossomos/metabolismo , Grânulos de Estresse , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Oxidativo , Mamíferos/metabolismoRESUMO
The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a hexanucleotide repeat expansion in C9orf72 (C9-HRE). While RNA and dipeptide repeats produced by C9-HRE disrupt nucleocytoplasmic transport, the proteins that become redistributed remain unknown. Here, we utilized subcellular fractionation coupled with tandem mass spectrometry and identified 126 proteins, enriched for protein translation and RNA metabolism pathways, which collectively drive a shift toward a more cytosolic proteome in C9-HRE cells. Among these was eRF1, which regulates translation termination and nonsense-mediated decay (NMD). eRF1 accumulates within elaborate nuclear envelope invaginations in patient induced pluripotent stem cell (iPSC) neurons and postmortem tissue and mediates a protective shift from protein translation to NMD-dependent mRNA degradation. Overexpression of eRF1 and the NMD driver UPF1 ameliorate C9-HRE toxicity in vivo. Our findings provide a resource for proteome-wide nucleocytoplasmic alterations across neurodegeneration-associated repeat expansion mutations and highlight eRF1 and NMD as therapeutic targets in C9orf72-associated ALS and/or FTD.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/genética , Proteínas de Drosophila/genética , Demência Frontotemporal/genética , Neurônios/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido/genética , Fatores de Terminação de Peptídeos/genética , RNA Mensageiro/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Animais , Proteína C9orf72/metabolismo , Fracionamento Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Demência Frontotemporal/metabolismo , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas , Membrana Nuclear , Terminação Traducional da Cadeia Peptídica/genética , Fatores de Terminação de Peptídeos/metabolismo , Biossíntese de Proteínas , Proteoma , Frações Subcelulares , Espectrometria de Massas em TandemRESUMO
Intracellular pathogens often exploit RAB functions to establish a safe haven in which to survive and proliferate. Ehrlichia chaffeensis, an obligatory intracellular bacterium, resides in specialized membrane-bound inclusions that have early endosome-like characteristics, e.g., resident RAB5 GTPase and RAB5 effectors, including VPS34 (the catalytic subunit of class III phosphatidylinositol 3-kinase), but the inclusions lack late endosomal or lysosomal markers. Within inclusions, Ehrlichia obtains host-derived nutrients by inducing RAB5-regulated autophagy using Ehrlichia translocated factor-1 deployed by its type IV secretion system. This manipulation of RAB5 by a bacterial molecule offers a simple strategy for Ehrlichia to avoid destruction in lysosomes and obtain nutrients, membrane components, and a homeostatic intra-host-cell environment in which to grow.
Assuntos
Morte Celular Autofágica , Ehrlichia chaffeensis/fisiologia , Ehrlichiose/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Sistemas de Secreção Tipo IV/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Ehrlichiose/patologia , Endossomos/metabolismo , Endossomos/microbiologia , Humanos , Lisossomos/metabolismo , Lisossomos/microbiologiaRESUMO
Survival of Ehrlichia chaffeensis depends on obligatory intracellular infection. One of the barriers to E. chaffeensis research progress has been the inability, using conventional techniques, to generate knock-out mutants for genes essential for intracellular infection. This study examined the use of Peptide Nucleic Acids (PNAs) technology to interrupt type IV secretion system (T4SS) effector protein expression in E. chaffeensis followed by intracellular complementation of the effector to determine its requirement for infection. Successful E. chaffeensis infection depends on the E. chaffeensis-specific T4SS protein effector, ehrlichial translocated factor-1 (Etf-1), which induces Rab5-regulated autophagy to provide host cytosolic nutrients required for E. chaffeensis proliferation. Etf-1 is also imported by host cell mitochondria where it inhibits host cell apoptosis to prolong its infection. We designed a PNA specific to Etf-1 and showed that the PNA bound to the target region of single-stranded Etf-1 RNA using a competitive binding assay. Electroporation of E. chaffeensis with this PNA significantly reduced Etf-1 mRNA and protein, and the bacteria's ability to induce host cell autophagy and infect host cells. Etf-1 PNA-mediated inhibition of ehrlichial Etf-1 expression and E. chaffeensis infection could be intracellularly trans-complemented by ectopic expression of Etf-1-GFP in host cells. These data affirmed the critical role of bacterial T4SS effector in host cell autophagy and E. chaffeensis infection, and demonstrated the use of PNA to analyze the gene functions of obligate intracellular bacteria.
Assuntos
Proteínas do Sistema Complemento/metabolismo , Ehrlichia chaffeensis/genética , Ehrlichia chaffeensis/metabolismo , Ehrlichiose/imunologia , Técnicas de Silenciamento de Genes , Interações Hospedeiro-Patógeno/imunologia , Ácidos Nucleicos Peptídicos/genética , Sistemas de Secreção Tipo IV/metabolismo , Apoptose , Autofagia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ehrlichia chaffeensis/imunologia , Ehrlichia chaffeensis/patogenicidade , Ehrlichiose/microbiologia , Regulação Bacteriana da Expressão Gênica , Células HEK293 , Humanos , Mitocôndrias/metabolismo , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , RNA Mensageiro/metabolismo , Células THP-1 , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing.