RESUMO
The bacteria causing Legionnaires' disease, Legionella pneumophila, replicate intracellularly within unique Legionella-containing vacuoles (LCVs). LCV formation involves a type IV secretion system (T4SS) that translocates effector proteins into host cells. We show that the T4SS effector RidL localizes to LCVs, supports intracellular bacterial growth, and alters retrograde trafficking, in which selected proteins are transported from endosomes to the Golgi. The retromer complex that mediates retrograde trafficking localizes to LCVs independently of RidL and restricts intracellular bacterial growth. RidL binds the Vps29 retromer subunit and the lipid PtdIns(3)P, which localizes retromer components to membranes. Additionally, specific retromer cargo receptors and sorting nexins that mediate protein capture and membrane remodeling preferentially localize to LCVs in the absence of ridL. Ectopic RidL production inhibits retrograde trafficking, and L. pneumophila blocks retrograde transport at endosome exit sites in a ridL-dependent manner. Collectively, these findings suggest that RidL inhibits retromer function to promote intracellular bacterial replication.
Assuntos
Proteínas de Bactérias/metabolismo , Legionella pneumophila/metabolismo , Doença dos Legionários/metabolismo , Doença dos Legionários/microbiologia , Vacúolos/microbiologia , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Endossomos/metabolismo , Humanos , Legionella pneumophila/genética , Camundongos , Transporte Proteico , Vacúolos/metabolismoRESUMO
The environmental bacterium Legionella pneumophila naturally parasitizes free-living amoebae. L. pneumophila is an opportunistic human pathogen that grows in macrophages, thus causing a life-threatening pneumonia termed Legionnaires' disease. The bacteria replicate intracellularly in environmental and immune phagocytes within a unique compartment, the Legionella-containing vacuole (LCV). Formation of LCVs is a complex and robust process involving >150 secreted bacterial effector proteins, which are believed to subvert host cell signaling and vesicle trafficking pathways. This unit describes a simple approach to purify intact LCVs from Dictyostelium discoideum amoebae. The method comprises a two-step purification protocol that includes immuno-magnetic separation by means of an antibody against an effector protein specifically binding to LCVs, followed by density gradient centrifugation. The use of D. discoideum producing a fluorescent LCV marker and fluorescently labeled L. pneumophila allow tracking the enrichment of LCVs by light microscopy.
Assuntos
Fracionamento Celular/métodos , Separação Imunomagnética/métodos , Legionella/isolamento & purificação , Vacúolos/microbiologia , Animais , Centrifugação com Gradiente de Concentração , Dictyostelium/citologia , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/microbiologia , Legionella/citologia , Legionella/crescimento & desenvolvimento , Legionella/fisiologia , Microscopia de FluorescênciaRESUMO
Phosphoinositide (PI) glycerolipids are key regulators of eukaryotic signal transduction, cytoskeleton architecture and membrane dynamics. The host cell PI metabolism is targeted by intracellular bacterial pathogens, which evolved intricate strategies to modulate uptake processes and vesicle trafficking pathways. Upon entering eukaryotic host cells, pathogenic bacteria replicate in distinct vacuoles or in the host cytoplasm. Vacuolar pathogens manipulate PI levels to mimic or modify membranes of subcellular compartments and thereby establish their replicative niche. Legionella pneumophila, Brucella abortus, Mycobacterium tuberculosis and Salmonella enterica translocate effector proteins into the host cell, some of which anchor to the vacuolar membrane via PIs or enzymatically turnover PIs. Cytoplasmic pathogens target PI metabolism at the plasma membrane, thus modulating their uptake and antiapoptotic signalling pathways. Employing this strategy, Shigella flexneri directly injects a PI-modifying effector protein, while Listeria monocytogenes exploits PI metabolism indirectly by binding to transmembrane receptors. Thus, regardless of the intracellular lifestyle of the pathogen, PI metabolism is critically involved in the interactions with host cells.
Assuntos
Bactérias/patogenicidade , Fosfatidilinositóis/metabolismo , Vacúolos/microbiologia , Bactérias/metabolismo , Fagossomos/microbiologia , Fosfatidilinositol 3-Quinases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transdução de SinaisRESUMO
The causative agent of Legionnaires disease, Legionella pneumophila, forms a replicative vacuole in phagocytes by means of the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system and translocated effector proteins, some of which subvert host GTP and phosphoinositide (PI) metabolism. The Icm/Dot substrate SidC anchors to the membrane of Legionella-containing vacuoles (LCVs) by specifically binding to phosphatidylinositol 4-phosphate (PtdIns(4)P). Using a nonbiased screen for novel L. pneumophila PI-binding proteins, we identified the Rab1 guanine nucleotide exchange factor (GEF) SidM/DrrA as the predominant PtdIns(4)P-binding protein. Purified SidM specifically and directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L. pneumophila Arf1 GEF RalF did not bind to any PIs. The PtdIns(4)P-binding domain of SidM was mapped to the 12-kDa C-terminal sequence, termed "P4M" (PtdIns4P binding of SidM/DrrA). The isolated P4M domain is largely helical and displayed higher PtdIns(4)P binding activity in the context of the alpha-helical, monomeric full-length protein. SidM constructs containing P4M were translocated by Icm/Dot-proficient L. pneumophila and localized to the LCV membrane, indicating that SidM anchors to PtdIns(4)P on LCVs via its P4M domain. An L. pneumophila DeltasidM mutant strain displayed significantly higher amounts of SidC on LCVs, suggesting that SidM and SidC compete for limiting amounts of PtdIns(4)P on the vacuole. Finally, RNA interference revealed that PtdIns(4)P on LCVs is specifically formed by host PtdIns 4-kinase IIIbeta. Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase IIIbeta to anchor the effectors SidC and SidM to LCVs.
Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte/química , Fatores de Troca do Nucleotídeo Guanina/química , Legionella pneumophila/química , Fosfatos de Fosfatidilinositol/química , Proteínas rab1 de Ligação ao GTP/química , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Drosophila , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Legionella pneumophila/patogenicidade , Doença dos Legionários/genética , Doença dos Legionários/metabolismo , Mutação , Mapeamento de Peptídeos , Fagócitos/metabolismo , Fagócitos/microbiologia , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Vacúolos/genética , Vacúolos/metabolismo , Vacúolos/microbiologia , Proteínas rab1 de Ligação ao GTP/genética , Proteínas rab1 de Ligação ao GTP/metabolismoRESUMO
Legionella pneumophila, the causative agent of Legionnaires' disease, replicates in macrophages and amoebae within 'Legionella-containing vacuoles' (LCVs), which communicate with the early secretory pathway and the endoplasmic reticulum. Formation of LCVs requires the bacterial Icm/Dot type IV secretion system. The Icm/Dot-translocated effector protein SidC selectively anchors to LCVs by binding the host lipid phosphatidylinositol-4-phosphate (PtdIns(4)P). Here, we describe a novel and simple approach to purify intact vacuoles formed by L. pneumophila within Dictyostelium discoideum by using magnetic immunoseparation with an antibody against SidC, followed by density gradient centrifugation. To monitor LCV purification by fluorescence microscopy, we used Dictyostelium producing the LCV marker calnexin-GFP and L. pneumophila labeled with the red fluorescent protein DsRed. A proteome analysis of purified LCVs by liquid chromatography coupled to tandem mass spectrometry revealed 566 host proteins, including known LCV components, such as the small GTPases Arf1, Rab1 and Rab7. Rab8, an endosomal regulator of the late secretory pathway originating from the trans Golgi network, and the endosomal GTPase Rab14 were identified as novel LCV components, which were found to be present on vacuoles harboring wild-type but not Icm/Dot-deficient L. pneumophila. Thus, LCVs also communicate with the late secretory and endosomal pathways. Depletion of Rab8 or Arf1 by RNA interference reduced the amount of SidC on LCVs, indicating that the GTPases promote the recruitment of Legionella effectors by regulating the level of PtdIns(4)P.
Assuntos
Endossomos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Legionella pneumophila/metabolismo , Proteoma/análise , Proteoma/metabolismo , Via Secretória , Vacúolos/enzimologia , Animais , Dictyostelium/metabolismo , Técnicas Imunológicas , Legionella pneumophila/genética , Magnetismo , Fagócitos/metabolismoRESUMO
Legionella pneumophila, the causative agent of Legionnaires' disease, replicates within a specific vacuole in amoebae and macrophages. To form these 'Legionella-containing vacuoles' (LCVs), the bacteria employ the Icm/Dot type IV secretion system and effector proteins, some of which anchor to the LCV membrane via the host glycolipid phosphatidylinositol 4-phosphate [PtdIns(4)P]. Here we analysed the role of inositol polyphosphate 5-phosphatases (IP5Ps) during L. pneumophila infections. Bacterial replication and LCV formation occurred more efficiently in Dictyostelium discoideum amoebae lacking the IP5P Dd5P4, a homologue of human OCRL1 (Oculocerebrorenal syndrome of Lowe), implicated in retrograde endosome to Golgi trafficking. The phenotype was complemented by Dd5P4 but not the catalytically inactive 5-phosphatase. Ectopically expressed Dd5P4 or OCRL1 localized to LCVs in D. discoideum via an N-terminal domain previously not implicated in membrane targeting, and OCRL1 was also identified on LCVs in macrophages. Dd5P4 was catalytically active on LCVs and accumulated on LCVs harbouring wild-type but not DeltaicmT mutant L. pneumophila. The N-terminal domain of OCRL1 bound L. pneumophila LpnE, a Sel1-like repeat protein involved in LCV formation, which localizes to LCVs and selectively binds PtdIns(3)P. Our results indicate that OCRL1 restricts intracellular growth of L. pneumophila and binds to LCVs in association with LpnE.
Assuntos
Proteínas de Bactérias/metabolismo , Dictyostelium/enzimologia , Dictyostelium/microbiologia , Legionella pneumophila/crescimento & desenvolvimento , Monoéster Fosfórico Hidrolases/metabolismo , Vacúolos/microbiologia , Fatores de Virulência/metabolismo , Animais , Linhagem Celular , Deleção de Genes , Teste de Complementação Genética , Inositol Polifosfato 5-Fosfatases , Macrófagos/enzimologia , Macrófagos/microbiologia , Camundongos , Monoéster Fosfórico Hidrolases/análise , Monoéster Fosfórico Hidrolases/genética , Ligação Proteica , Vacúolos/químicaRESUMO
Legionella pneumophila, the causative agent of Legionnaires' disease, uses the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system to establish within amoebae and macrophages an endoplasmic reticulum (ER)-derived replication-permissive compartment, the Legionella-containing vacuole (LCV). The Icm/Dot substrate SidC and its paralogue SdcA anchor to LCVs via phosphatidylinositol-4 phosphate [PtdIns(4)P]. Here we identify the unique 20 kDa PtdIns(4)P-binding domain of SidC, which upon heterologous expression in Dictyostelium binds to LCVs and thus is useful as a PtdIns(4)P-specific probe. LCVs harbouring L. pneumophilaDeltasidC-sdcA mutant bacteria recruit ER and ER-derived vesicles less efficiently and carry endosomal but not lysosomal markers. The phenotypes are complemented by supplying sidC on a plasmid. L. pneumophilaDeltasidC-sdcA grows at wild-type rate in calnexin-negative LCVs, suggesting that communication with the ER is dispensable for establishing a replicative compartment. The amount of SidC and calnexin is directly proportional on isolated LCVs, and in a cell-free system, the recruitment of calnexin-positive vesicles to LCVs harbouring DeltasidC-sdcA mutant bacteria is impaired. Beads coated with purified SidC or its 70 kDa N-terminal fragment recruit ER vesicles in Dictyostelium and macrophage lysates. Our results establish SidC as an L. pneumophila effector protein, which anchors to PtdIns(4)P on LCVs and recruits ER vesicles to a replication-permissive vacuole.
Assuntos
Proteínas de Bactérias/metabolismo , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/microbiologia , Legionella pneumophila/patogenicidade , Fatores de Virulência/metabolismo , Proteínas de Bactérias/genética , Calnexina/análise , Vesículas Citoplasmáticas/química , Vesículas Citoplasmáticas/ultraestrutura , Deleção de Genes , Teste de Complementação Genética , Humanos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Fatores de Virulência/genéticaRESUMO
Environmental bacteria are constantly threatened by bacterivorous predators such as free-living protozoa and nematodes. In the course of their coevolution with environmental predators, some bacteria developed sophisticated defence mechanisms, including the secretion of toxins, or the capacity to avoid lysosomal killing and to replicate intracellularly within protozoa. To analyse the interactions with bacterial pathogens on a molecular, cellular or organismic level, protozoa and other non-mammalian hosts are increasingly used. These include amoebae, as well as genetically tractable hosts, such as the social amoeba Dictyostelium discoideum, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. Using these hosts, the virulence mechanisms of opportunistic pathogenic bacteria such as Legionella, Mycobacterium, Pseudomonas or Vibrio were found to be not only relevant for the interactions of the bacteria with protozoa, nematodes and insect phagocytes, but also with mammalian hosts including humans. Thus, non-mammalian model hosts provide valuable insight into the pathogenesis of environmental bacteria.
Assuntos
Amoeba/microbiologia , Caenorhabditis elegans/microbiologia , Drosophila melanogaster/microbiologia , Bactérias Gram-Negativas/patogenicidade , Bactérias Gram-Positivas/patogenicidade , Animais , Infecções Bacterianas/microbiologia , Infecções Bacterianas/patologia , Modelos Animais de Doenças , Humanos , Comportamento PredatórioRESUMO
The causative agent of Legionnaires' disease, Legionella pneumophila, employs the intracellular multiplication (Icm)/defective organelle trafficking (Dot) type IV secretion system (T4SS) to upregulate phagocytosis and to establish a replicative vacuole in amoebae and macrophages. Legionella-containing vacuoles (LCVs) do not fuse with endosomes but recruit early secretory vesicles. Here we analyze the role of host cell phosphoinositide (PI) metabolism during uptake and intracellular replication of L. pneumophila. Genetic and pharmacological evidence suggests that class I phosphatidylinositol(3) kinases (PI3Ks) are dispensable for phagocytosis of wild-type L. pneumophila but inhibit intracellular replication of the bacteria and participate in the modulation of the LCV. Uptake and degradation of an icmT mutant strain lacking a functional Icm/Dot transporter was promoted by PI3Ks. We identified Icm/Dot-secreted proteins which specifically bind to phosphatidylinositol(4) phosphate (PI(4)P) in vitro and preferentially localize to LCVs in the absence of functional PI3Ks. PI(4)P was found to be present on LCVs using as a probe either an antibody against PI(4)P or the PH domain of the PI(4)P-binding protein FAPP1 (phosphatidylinositol(4) phosphate adaptor protein-1). Moreover, the presence of PI(4)P on LCVs required a functional Icm/Dot T4SS. Our results indicate that L. pneumophila modulates host cell PI metabolism and exploits the Golgi lipid second messenger PI(4)P to anchor secreted effector proteins to the LCV.
Assuntos
Proteínas de Bactérias/metabolismo , Legionella pneumophila/fisiologia , Fosfatos de Fosfatidilinositol/fisiologia , Vacúolos/metabolismo , Animais , Proteínas de Bactérias/genética , Biomarcadores/metabolismo , Linhagem Celular , Dictyostelium/crescimento & desenvolvimento , Humanos , Legionella pneumophila/crescimento & desenvolvimento , Camundongos , Dados de Sequência Molecular , Mutação , Organelas/metabolismo , Fagocitose/fisiologia , Fosfatidilinositol 3-Quinases/deficiência , Fosfatidilinositol 3-Quinases/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , Transporte Proteico , Distribuição Tecidual , Vacúolos/microbiologiaRESUMO
Four genes coding for small heat shock proteins (sHsps) were identified in the genome sequence of Agrobacterium tumefaciens, one on the circular chromosome (hspC), one on the linear chromosome (hspL), and two on the pAT plasmid (hspAT1 and hspAT2). Induction of sHsps at elevated temperatures was revealed by immunoblot analyses. Primer extension experiments and translational lacZ fusions demonstrated that expression of the pAT-derived genes and hspL is controlled by temperature in a regulon-specific manner. While the sHsp gene on the linear chromosome turned out to be regulated by RpoH (sigma32), both copies on pAT were under the control of highly conserved ROSE (named for repression of heat shock gene expression) sequences in their 5' untranslated region. Secondary structure predictions of the corresponding mRNA strongly suggest that it represses translation at low temperatures by masking the Shine-Dalgarno sequence. The hspC gene was barely expressed (if at all) and not temperature responsive.
Assuntos
Agrobacterium tumefaciens/fisiologia , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/metabolismo , Replicon , Elementos de Resposta/genética , Regiões 5' não Traduzidas/genética , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas de Choque Térmico/genética , Resposta ao Choque Térmico , Temperatura Alta , Dados de Sequência Molecular , Elementos de Resposta/fisiologia , Fator sigma/genética , Fator sigma/metabolismo , Transcrição GênicaRESUMO
Thermoresponsive structures in the 5'-untranslated region of mRNA are known to control translation of heat shock and virulence genes. Expression of many rhizobial heat shock genes is regulated by a conserved sequence element called ROSE for repression of heat shock gene expression. This cis-acting, untranslated mRNA is thought to prevent ribosome access at low temperature through an extended secondary structure, which partially melts when the temperature rises. We show here by a series of in vivo and in vitro approaches that ROSE is a sensitive thermometer responding in the physiologically relevant temperature range between 30 and 40 degrees C. Point mutations predicted to disrupt base pairing enhanced expression at 30 degrees C. Compensatory mutations restored repression, emphasizing the importance of secondary structures in the sensory RNA. Only moderate inducibility of a 5'-truncated ROSE variant suggests that interactions between individual stem loops coordinate temperature sensing. In the presence of a complementary oligonucleotide, the functionally important stem loop of ROSE was rendered susceptible to RNase H treatment at heat shock temperatures. Since major structural rearrangements were not observed during UV and CD spectroscopy, subtle structural changes involving the Shine-Dalgarno sequence are proposed to mediate translational control. Temperature perception by the sensory RNA is an ordered process that most likely occurs without the aid of accessory factors.