RESUMO
Lymphatic vascular development establishes embryonic and adult tissue fluid balance and is integral in disease. In diverse vertebrate organs, lymphatic vessels display organotypic function and develop in an organ-specific manner. In all settings, developmental lymphangiogenesis is considered driven by vascular endothelial growth factor (VEGF) receptor-3 (VEGFR3), whereas a role for VEGFR2 remains to be fully explored. Here, we define the zebrafish Vegf/Vegfr code in receptor binding studies. We find that while Vegfd directs craniofacial lymphangiogenesis, it binds Kdr (a VEGFR2 homolog) but surprisingly, unlike in mammals, does not bind Flt4 (VEGFR3). Epistatic analyses and characterization of a kdr mutant confirm receptor-binding analyses, demonstrating that Kdr is indispensible for rostral craniofacial lymphangiogenesis, but not caudal trunk lymphangiogenesis, in which Flt4 is central. We further demonstrate an unexpected yet essential role for Kdr in inducing lymphatic endothelial cell fate. This work reveals evolutionary divergence in the Vegf/Vegfr code that uncovers spatially restricted mechanisms of developmental lymphangiogenesis.
Assuntos
Células Endoteliais/metabolismo , Evolução Molecular , Linfangiogênese , Fator C de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Células HEK293 , Humanos , Ligantes , Camundongos , Ligação Proteica , Proteólise , Reprodutibilidade dos Testes , Fator C de Crescimento do Endotélio Vascular/química , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismoRESUMO
The human pathogen Legionella pneumophila must evade host cell death signaling to enable replication in lung macrophages and to cause disease. After bacterial growth, however, L. pneumophila is thought to induce apoptosis during egress from macrophages. The bacterial effector protein, SidF, has been shown to control host cell survival and death by inhibiting pro-apoptotic BNIP3 and BCL-RAMBO signaling. Using live-cell imaging to follow the L. pneumophila-macrophage interaction, we now demonstrate that L. pneumophila evades host cell apoptosis independent of SidF. In the absence of SidF, L. pneumophila was able to replicate, cause loss of mitochondria membrane potential, kill macrophages, and establish infections in lungs of mice. Consistent with this, deletion of BNIP3 and BCL-RAMBO did not affect intracellular L. pneumophila replication, macrophage death rates, and in vivo bacterial virulence. Abrogating mitochondrial cell death by genetic deletion of the effectors of intrinsic apoptosis, BAX, and BAK, or the regulator of mitochondrial permeability transition pore formation, cyclophilin-D, did not affect bacterial growth or the initial killing of macrophages. Loss of BAX and BAK only marginally limited the ability of L. pneumophila to efficiently kill all macrophages over extended periods. L. pneumophila induced killing of macrophages was delayed in the absence of capsase-11 mediated pyroptosis. Together, our data demonstrate that L. pneumophila evades host cell death responses independently of SidF during replication and can induce pyroptosis to kill macrophages in a timely manner.
Assuntos
Proteínas de Bactérias/genética , Flagelina/genética , Legionella pneumophila/fisiologia , Doença dos Legionários/microbiologia , Macrófagos/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Bactérias/metabolismo , Morte Celular , Sobrevivência Celular , Feminino , Flagelina/metabolismo , Deleção de Genes , Interações Hospedeiro-Patógeno , Doença dos Legionários/metabolismo , Macrófagos/microbiologia , Proteínas de Membrana/metabolismo , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Fatores de Virulência/genéticaRESUMO
Vascular endothelial growth factors (VEGFs) control angiogenesis and lymphangiogenesis during development and in pathological conditions. In the zebrafish trunk, Vegfa controls the formation of intersegmental arteries by primary angiogenesis and Vegfc is essential for secondary angiogenesis, giving rise to veins and lymphatics. Vegfd has been largely thought of as dispensable for vascular development in vertebrates. Here, we generated a zebrafish vegfd mutant by genome editing. vegfd mutants display significant defects in facial lymphangiogenesis independent of vegfc function. Strikingly, we find that vegfc and vegfd cooperatively control lymphangiogenesis throughout the embryo, including during the formation of the trunk lymphatic vasculature. Interestingly, we find that vegfd and vegfc also redundantly drive artery hyperbranching phenotypes observed upon depletion of Flt1 or Dll4. Epistasis and biochemical binding assays suggest that, during primary angiogenesis, Vegfd influences these phenotypes through Kdr (Vegfr2) rather than Flt4 (Vegfr3). These data demonstrate that, rather than being dispensable during development, Vegfd plays context-specific indispensable and also compensatory roles during both blood vessel angiogenesis and lymphangiogenesis.
Assuntos
Linfangiogênese/fisiologia , Neovascularização Fisiológica/fisiologia , Fator D de Crescimento do Endotélio Vascular/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/fisiologia , Animais , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Linfangiogênese/genética , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Modelos Biológicos , Mutagênese , Neovascularização Fisiológica/genética , Deleção de Sequência , Transdução de Sinais , Regulação para Cima , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/fisiologia , Fator D de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Human pathogenic Legionella replicate in alveolar macrophages and cause a potentially lethal form of pneumonia known as Legionnaires' disease(1). Here, we have identified a host-directed therapeutic approach to eliminate intracellular Legionella infections. We demonstrate that the genetic deletion, or pharmacological inhibition, of the host cell pro-survival protein BCL-XL induces intrinsic apoptosis of macrophages infected with virulent Legionella strains, thereby abrogating Legionella replication. BCL-XL is essential for the survival of Legionella-infected macrophages due to bacterial inhibition of host-cell protein synthesis, resulting in reduced levels of the short-lived, related BCL-2 pro-survival family member, MCL-1. Consequently, a single dose of a BCL-XL-targeted BH3-mimetic therapy, or myeloid cell-restricted deletion of BCL-XL, limits Legionella replication and prevents lethal lung infections in mice. These results indicate that repurposing BH3-mimetic compounds, originally developed to induce cancer cell apoptosis, may have efficacy in treating Legionnaires' and other diseases caused by intracellular microbes.
Assuntos
Apoptose , Legionella/crescimento & desenvolvimento , Macrófagos Alveolares/microbiologia , Macrófagos Alveolares/fisiologia , Proteína bcl-X/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Deleção de Genes , Legionelose/prevenção & controle , Camundongos , Proteína bcl-X/genéticaRESUMO
The translocation of effector proteins by the Dot/Icm type IV secretion system is central to the ability of Legionella pneumophila to persist and replicate within eukaryotic cells. The subcellular localization of translocated Dot/Icm proteins in host cells provides insight into their function. Through co-staining with host cell markers, effector proteins may be localized to specific subcellular compartments and membranes, which frequently reflects their host cell target and mechanism of action. In this chapter, we describe protocols to (1) localize effector proteins within cells by ectopic expression using green fluorescent protein fusions and (2) localize effector proteins within infected cells using epitope-tagged effector proteins and immuno-fluorescence microscopy.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Legionella/metabolismo , Proteínas de Bactérias/genética , Clonagem Molecular , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Legionella/genética , Legionella pneumophila/metabolismo , Microscopia de Fluorescência , Plasmídeos/genética , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Legionella pneumophila is a ubiquitous inhabitant of environmental water reservoirs. The bacteria infect a wide variety of protozoa and, after accidental inhalation, human alveolar macrophages, which can lead to severe pneumonia. The capability to thrive in phagocytic hosts is dependent on the Dot/Icm type IV secretion system (T4SS), which translocates multiple effector proteins into the host cell. In this study, we determined the draft genome sequence of L. pneumophila strain 130b (Wadsworth). We found that the 130b genome encodes a unique set of T4SSs, namely, the Dot/Icm T4SS, a Trb-1-like T4SS, and two Lvh T4SS gene clusters. Sequence analysis substantiated that a core set of 107 Dot/Icm T4SS effectors was conserved among the sequenced L. pneumophila strains Philadelphia-1, Lens, Paris, Corby, Alcoy, and 130b. We also identified new effector candidates and validated the translocation of 10 novel Dot/Icm T4SS effectors that are not present in L. pneumophila strain Philadelphia-1. We examined the prevalence of the new effector genes among 87 environmental and clinical L. pneumophila isolates. Five of the new effectors were identified in 34 to 62% of the isolates, while less than 15% of the strains tested positive for the other five genes. Collectively, our data show that the core set of conserved Dot/Icm T4SS effector proteins is supplemented by a variable repertoire of accessory effectors that may partly account for differences in the virulences and prevalences of particular L. pneumophila strains.