RESUMEN
Listeria monocytogenes is an intracellular bacterial pathogen that replicates rapidly in the cytosol of host cells during acute infection. Surprisingly, these bacteria were found to occupy vacuoles in liver granuloma macrophages during persistent infection of severe combined immunodeficient (SCID) mice. Here we show that L. monocytogenes can replicate in vacuoles within macrophages. In livers of SCID mice infected for 21 days, we observed bacteria in large LAMP1(+) compartments that we termed spacious Listeria-containing phagosomes (SLAPs). SLAPs were also observed in vitro, and were found to be non-acidic and non-degradative compartments that are generated in an autophagy-dependent manner. The replication rate of bacteria in SLAPs was found to be reduced compared to the rate of those in the cytosol. Listeriolysin O (LLO, encoded by hly), a pore-forming toxin essential for L. monocytogenes virulence, was necessary and sufficient for SLAP formation. A L. monocytogenes mutant with low LLO expression was impaired for phagosome escape but replicated slowly in SLAPs over a 72 h period. Therefore, our studies reveal a role for LLO in promoting L. monocytogenes replication in vacuoles and suggest a mechanism by which this pathogen can establish persistent infection in host macrophages.
Asunto(s)
Toxinas Bacterianas/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/metabolismo , Listeria monocytogenes/crecimiento & desarrollo , Listeria monocytogenes/patogenicidad , Macrófagos/citología , Macrófagos/microbiología , Vacuolas/microbiología , Animales , Autofagia , Toxinas Bacterianas/genética , Enfermedad Crónica , Proteínas de Choque Térmico/genética , Proteínas Hemolisinas/genética , Listeria monocytogenes/metabolismo , Listeriosis/microbiología , Listeriosis/patología , Hígado/microbiología , Proteínas de Membrana de los Lisosomas/metabolismo , Ratones , Ratones SCID , Fagosomas/metabolismo , Fagosomas/microbiología , Vacuolas/metabolismo , VirulenciaRESUMEN
In order to ensure the safety of vaccines produced on avian cells, rigorous testing for the absence of avian retroviruses must be performed. Current methods used to detect avian retroviruses often exhibit a high invalid-test/false-positive rate, rely on hard-to-secure reagents, and/or have readouts that are difficult to standardize. Herein, we describe the development and validation of two consistent and sensitive methods for the detection of avian retroviruses in vaccines: viral amplification on DF-1 cells followed by immunostaining for the detection of avian leukosis virus (ALV) and viral amplification on DF-1 cells followed by fluorescent product-enhanced reverse transcriptase (F-PERT) for the detection of all avian retroviruses. Both assays share an infectivity stage on DF-1 cells followed by a different endpoint readout depending on the retrovirus to be detected. Validation studies demonstrated a limit of detection of one 50% cell culture infectious dose (CCID(50))/ml for retrovirus in a 30-ml test inoculum volume for both methods, which was as sensitive as a classical method used in the vaccine industry, namely, viral amplification on primary chicken embryo fibroblasts followed by the complement fixation test for avian leukosis virus (COFAL). Furthermore, viral amplification on DF-1 cells followed by either immunostaining or F-PERT demonstrated a sensitivity that exceeds the regulatory requirements for detection of ALV strains. A head-to-head comparison of the two endpoint methods showed that viral amplification on DF-1 cells followed by F-PERT is a suitable method to be used as a stand-alone test to ensure that vaccine preparations are free from infectious avian retroviruses.
Asunto(s)
Virus de la Leucosis Aviar/aislamiento & purificación , Contaminación de Medicamentos , Vacunas , Animales , Virus de la Leucosis Aviar/genética , Virus de la Leucosis Aviar/crecimiento & desarrollo , Línea Celular , Embrión de Pollo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Replicación ViralRESUMEN
MEI-1, the catalytic subunit of the Caenorhabditis elegans "katanin" microtubule-severing complex, is required for meiotic spindle formation. However, MEI-1 must be inactivated after the completion of meiosis to allow formation of the first mitotic spindle. Recent work demonstrated that post-meiotic MEI-1 undergoes ubiquitin-dependent degradation mediated by two independent pathways. Here we describe another level of MEI-1 regulation involving the protein phosphatase 4 (PP4) complex. The PP4 R1 regulatory subunit protein phosphatase four regulatory subunit 1 (ppfr-1) was identified in an RNA interference (RNAi) screen for suppressors of a mei-1(gf) allele that is refractory to post-meiotic degradation. RNAi to the PP4 catalytic subunit PPH-4.1 or to the alpha4 regulatory PPFR-4 also suppressed lethality of ectopic MEI-1. These results suggest that PP4(+) activates MEI-1, and therefore loss of PP4 decreases ectopic MEI-1(gf) activity. PPH-4.1 and MEI-1 co-immunoprecipitate with one another, indicating that the PP4 complex likely regulates MEI-1 activity directly rather than through an intermediate. The ppfr-1 mutant has subtle meiotic defects indicating that PPFR-1 also regulates MEI-1 during meiosis. MBK-2 is the only kinase known to phosphorylate MEI-1 and triggers post-meiotic MEI-1 degradation. However, genetic interactions between PP4 and mbk-2 were not consistent with an antagonistic relationship between the phosphatase and kinase. Additionally, reducing PP4 in mei-1(gf) did not change the level or localization of post-meiotic MEI-1. Thus, by making use of a genetic background where MEI-1 is ectopically expressed, we have uncovered a third mechanism of MEI-1 regulation, one based on phosphorylation but independent of degradation. The redundant regulatory pathways likely contribute in different ways to the rapid and precise post-meiotic inactivation of MEI-1 microtubule-severing activity.
Asunto(s)
Caenorhabditis elegans/citología , Caenorhabditis elegans/enzimología , Microtúbulos/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Técnicas de Silenciamiento del Gen , Meiosis , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Fosforilación , Subunidades de Proteína/deficiencia , Subunidades de Proteína/genéticaRESUMEN
Recent studies have suggested the existence of innate host surveillance systems for the detection of bacteria in the cytosol of mammalian cells. The molecular details of how bacteria are recognized in the cytosol, however, remain unclear. Here we examined the fate of Salmonella typhimurium, a gram-negative bacterial pathogen that can infect a variety of hosts, in the cytosol of mammalian cells. These bacteria typically occupy a membrane bound compartment, the Salmonella-containing vacuole (SCV), in host cells. We show that some wild-type bacteria escape invasion vacuoles and are released into the cytosol. Subsequently, polyubiquitinated proteins accumulate on the bacterial surface, a response that was witnessed in several cell types. In macrophages but not epithelial cells, the proteasome was observed to undergo a dramatic subcellular relocalization and become associated with the surface of bacteria in the cytosol. Proteasome inhibition promoted replication of S. typhimurium in the cytosol of both cell types, in part through destabilization of the SCV. Surprisingly, the cytosol-adapted pathogen Listeria monocytogenes avoided recognition by the ubiquitin system by using actin-based motility. Our findings indicate that the ubiquitin system plays a major role in the recognition of bacterial pathogens in the cytosol of mammalian cells.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Citosol/microbiología , Macrófagos/inmunología , Complejos Multienzimáticos/metabolismo , Salmonella typhimurium/metabolismo , Ubiquitinas/metabolismo , Actinas/metabolismo , Animales , Células Cultivadas , Cisteína Endopeptidasas/inmunología , Citosol/inmunología , Técnica del Anticuerpo Fluorescente , Células HeLa , Hipocampo/citología , Humanos , Listeria monocytogenes/inmunología , Listeria monocytogenes/metabolismo , Listeria monocytogenes/ultraestructura , Microscopía Confocal , Microscopía Electrónica , Complejos Multienzimáticos/inmunología , Complejo de la Endopetidasa Proteasomal , Ratas , Salmonella typhimurium/inmunología , Salmonella typhimurium/ultraestructura , Ubiquitinas/inmunología , Vacuolas/metabolismoRESUMEN
Autophagy mediates the degradation of cytoplasmic components in eukaryotic cells and plays a key role in immunity. The mechanism of autophagosome formation is not clear. Here we examined two potential membrane sources for antibacterial autophagy: the ER and mitochondria. DFCP1, a marker of specialized ER domains known as 'omegasomes,' associated with Salmonella-containing autophagosomes via its PtdIns(3)P and ER-binding domains, while a mitochondrial marker (cytochrome b5-GFP) did not. Rab1 also localized to autophagosomes, and its activity was required for autophagosome formation, clearance of protein aggregates and peroxisomes, and autophagy of Salmonella. Overexpression of Rab1 enhanced antibacterial autophagy. The role of Rab1 in antibacterial autophagy was independent of its role in ER-to-Golgi transport. Our data suggest that antibacterial autophagy occurs at omegasomes and reveal that the Rab1 GTPase plays a crucial role in mammalian autophagy.
Asunto(s)
Autofagia , Retículo Endoplásmico/enzimología , Membranas Intracelulares/enzimología , Fosfatos de Fosfatidilinositol/metabolismo , Salmonella typhimurium/inmunología , Proteínas de Unión al GTP rab1/metabolismo , Animales , Proteínas Portadoras/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Membranas Intracelulares/efectos de los fármacos , Ratones , Peroxisomas/efectos de los fármacos , Peroxisomas/metabolismo , Fagosomas/microbiología , Estructura Cuaternaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Sirolimus/farmacología , Proteínas Ubiquitinadas/químicaRESUMEN
Autophagy mediates the degradation of cytoplasmic contents in the lysosome and plays a significant role in immunity. Lipid second messengers have previously been implicated in the regulation of autophagy. Here, we demonstrate a signaling role for diacylglycerol (DAG) in antibacterial autophagy. DAG production was necessary for efficient autophagy of Salmonella, and its localization to bacteria-containing phagosomes preceded autophagy. The actions of phospholipase D and phosphatidic acid phosphatase were required for DAG generation and autophagy. Furthermore, the DAG-responsive delta isoform of protein kinase C was required, as were its downstream targets JNK and NADPH oxidase. Previous studies have revealed a role for the ubiquitin-binding adaptor molecules p62 and NDP52 in autophagy of S. Typhimurium. We observed bacteria-containing autophagosomes colocalizing individually with either DAG or ubiquitinated proteins, indicating that both signals can act independently to promote antibacterial autophagy. These findings reveal an important role for DAG-mediated PKC function in mammalian antibacterial autophagy.
Asunto(s)
Autofagia/fisiología , Diglicéridos/fisiología , Interacciones Huésped-Patógeno , Infecciones por Salmonella/inmunología , Salmonella typhimurium/fisiología , Animales , Línea Celular , Humanos , Fosfatidato Fosfatasa/fisiología , Fosfolipasa D/fisiología , Proteína Quinasa C/fisiología , Transducción de SeñalRESUMEN
Autophagy is an important component of the mammalian innate immune system and is able to specifically target intracellular bacterial pathogens. Salmonella enterica serovar Typhimurium is an intracellular pathogen that causes gastroenteritis in humans. Autophagy has been shown to target S. Typhimurium during in vitro infection of mammalian cultured cells and protects the cytosol of these cells from bacterial colonization. Here we discuss autophagic sequestration of S. Typhimurium and how it can be used as a model system to study the mechanisms of autophagy.
Asunto(s)
Autofagia/fisiología , Salmonella typhimurium/inmunología , Animales , Proteínas Relacionadas con la Autofagia , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Células Cultivadas , Enfermedad de Crohn/metabolismo , Células HeLa , Humanos , Cinética , Ratones , Modelos Biológicos , Salmonelosis Animal/inmunología , Salmonelosis Animal/metabolismo , Salmonella typhimurium/crecimiento & desarrollo , Salmonella typhimurium/metabolismoRESUMEN
Autophagy restricts the growth of a variety of intracellular pathogens. However, cytosol-adapted pathogens have evolved ways to evade restriction by this innate immune mechanism. Listeria monocytogenes is a Gram-positive bacterial pathogen that utilizes a cholesterol-dependent pore-forming toxin, listeriolysin O (LLO), to escape from the phagosome. Autophagy targets L. monocytogenes in LLO-damaged phagosomes and also in the cytosol under some experimental conditions. However, this bacterium has evolved multiple mechanisms to evade restriction by autophagy, including actin-based motility in the cytosol and an as yet undefined mechanism mediated by bacterial phospholipases C (PLCs). A population of L. monocytogenes with inefficient LLO activity forms Spacious Listeria-containing Phagosomes (SLAPs), which are autophagosome-like compartments that do not mature, allowing slow bacterial growth within enlarged vesicles. SLAPs may represent a stalemate between bacterial LLO action and the host autophagy system, resulting in persistent infection.
Asunto(s)
Autofagia/fisiología , Listeria monocytogenes/fisiología , Macrófagos/microbiología , Actinas/fisiología , Animales , Proteínas Bacterianas/fisiología , Toxinas Bacterianas , Citosol/fisiología , Proteínas de Choque Térmico/fisiología , Proteínas Hemolisinas/fisiología , Listeriosis/metabolismo , Listeriosis/microbiología , Macrófagos/fisiología , Fagosomas/fisiología , Fosfolipasas de Tipo C/fisiologíaRESUMEN
Listeria monocytogenes is an intracellular pathogen that is able to colonize the cytosol of macrophages. Here we examined the interaction of this pathogen with autophagy, a host cytosolic degradative pathway that constitutes an important component of innate immunity towards microbial invaders. L. monocytogenes infection induced activation of the autophagy system in macrophages. At 1 h post infection (p.i.), a population of intracellular bacteria ( approximately 37%) colocalized with the autophagy marker LC3. These bacteria were within vacuoles and were targeted by autophagy in an LLO-dependent manner. At later stages in infection (by 4 h p.i.), the majority of L. monocytogenes escaped into the cytosol and rapidly replicated. At these times, less than 10% of intracellular bacteria colocalized with LC3. We found that ActA expression was sufficient to prevent autophagy of bacteria in the cytosol of macrophages. Surprisingly, ActA expression was not strictly necessary, indicating that other virulence factors were involved. Accordingly, we also found a role for the bacterial phospholipases, PI-PLC and PC-PLC, in autophagy evasion, as bacteria lacking phospholipase expression were targeted by autophagy at later times in infection. Together, our results demonstrate that L. monocytogenes utilizes multiple mechanisms to avoid destruction by the autophagy system during colonization of macrophages.
Asunto(s)
Autofagia/fisiología , Listeria monocytogenes/patogenicidad , Macrófagos/microbiología , Macrófagos/fisiología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Toxinas Bacterianas , Línea Celular , Genes Bacterianos , Proteínas de Choque Térmico/fisiología , Proteínas Hemolisinas/fisiología , Listeria monocytogenes/genética , Listeria monocytogenes/fisiología , Macrófagos/ultraestructura , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Ratones , Microscopía Electrónica de Transmisión , Modelos Biológicos , Mutación , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/fisiología , Fosfolipasas de Tipo C/fisiología , VirulenciaRESUMEN
Autophagy is responsible for the degradation of cytosolic components within eukaryotic cells. Interestingly, autophagy also appears to play a role in recognizing invading intracellular pathogens. Salmonella enterica serovar Typhimurium (S. Typhimurium) is an intracellular pathogen that normally resides and replicates within the Salmonella-containing vacuole (SCV). However, during in vitro infection a population of S. Typhimurium damage and escape from the SCV to enter the cytosol. We have observed that some intracellular S. Typhimurium are recognized by autophagy under in vitro infection conditions. Immunofluorescence studies revealed that autophagy recognizes the population of S. Typhimurium within damaged SCVs early after infection. The consequences of autophagic recognition of S. Typhimurium are still being elucidated, though a restrictive effect on intracellular bacterial replication has been demonstrated. Results of our in vitro infection studies are consistent with autophagy playing a role in cellular defense against S. Typhimurium that become exposed to the cytosol.
Asunto(s)
Autofagia/fisiología , Salmonella typhimurium/patogenicidad , Vacuolas/microbiología , Animales , Humanos , Modelos Biológicos , Infecciones por Salmonella/microbiología , Salmonelosis Animal/microbiologíaRESUMEN
Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen that causes disease in a variety of hosts. S. Typhimurium actively invade host cells and typically reside within a membrane-bound compartment called the Salmonella-containing vacuole (SCV). The bacteria modify the fate of the SCV using two independent type III secretion systems (TTSS). TTSS are known to damage eukaryotic cell membranes and S. Typhimurium has been suggested to damage the SCV using its Salmonella pathogenicity island (SPI)-1 encoded TTSS. Here we show that this damage gives rise to an intracellular bacterial population targeted by the autophagy system during in vitro infection. Approximately 20% of intracellular S. Typhimurium colocalized with the autophagy marker GFP-LC3 at 1 h postinfection. Autophagy of S. Typhimurium was dependent upon the SPI-1 TTSS and bacterial protein synthesis. Bacteria targeted by the autophagy system were often associated with ubiquitinated proteins, indicating their exposure to the cytosol. Surprisingly, these bacteria also colocalized with SCV markers. Autophagy-deficient (atg5-/-) cells were more permissive for intracellular growth by S. Typhimurium than normal cells, allowing increased bacterial growth in the cytosol. We propose a model in which the host autophagy system targets bacteria in SCVs damaged by the SPI-1 TTSS. This serves to retain intracellular S. Typhimurium within vacuoles early after infection to protect the cytosol from bacterial colonization. Our findings support a role for autophagy in innate immunity and demonstrate that Salmonella infection is a powerful model to study the autophagy process.
Asunto(s)
Citosol/microbiología , Infecciones por Salmonella/patología , Salmonella/fisiología , Animales , Autofagia , Membrana Celular/metabolismo , Citosol/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Fibroblastos/metabolismo , Fibroblastos/microbiología , Células HeLa , Humanos , Ratones , Microscopía Confocal , Microscopía Fluorescente , Modelos Biológicos , Plásmidos/metabolismo , Salmonella/metabolismo , Factores de Tiempo , Transfección , Vacuolas/metabolismoRESUMEN
Salmonella enterica serovar Typhimurium utilizes a type III secretion system (TTSS) encoded on Salmonella pathogenicity island-2 (SPI2) to promote intracellular replication during infection, but little is known about the molecular function of SPI2-translocated effectors and how they contribute to this process. SseJ is a SPI2 TTSS effector protein that is homologous to enzymes called glycerophospholipid-cholesterol acyltransferases and, following translocation, localizes to the Salmonella-containing vacuole and Salmonella-induced filaments. Full virulence requires SseJ, as sseJ null mutants exhibit decreased replication in cultured cells and host tissues. This work demonstrates that SseJ is an enzyme with deacylase activity in vitro and identifies three active-site residues. Catalytic SseJ mutants display wild-type translocation and subcellular localization but fail to complement the virulence defect of an sseJ null mutant. In contrast to the wild type, SseJ catalytic mutants fail to down regulate Salmonella-induced filament formation and fail to restore the sifA null mutant phenotype of loss of phagosomal membrane to sifA sseJ null double mutants, suggesting that wild-type SseJ modifies the vacuolar membrane. This is the first demonstration of an enzymatic activity for a SPI2 effector protein and provides support for the hypothesis that the deacylation of lipids on the Salmonella-containing vacuole membrane is important to bacterial pathogenesis.
Asunto(s)
Aciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/metabolismo , Salmonella typhimurium/enzimología , Salmonella typhimurium/patogenicidad , Aciltransferasas/análisis , Aciltransferasas/genética , Animales , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Dominio Catalítico , Femenino , Prueba de Complementación Genética , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Transporte de Proteínas , Vacuolas/enzimología , VirulenciaRESUMEN
Salmonella enterica serovar Typhimurium has the fascinating ability to form tubular structures known as Salmonella-induced filaments (Sifs) in host cells. Here, we show that the prevalence of the Sif phenotype in HeLa cells is affected by host cell density, growth, and the multiplicity of infection. Sif formation was observed in cells that displayed rapid intracellular bacterial replication and was found to be dynamic, being maximal 8 to 10 h postinfection and declining thereafter. The virulence factors SpvB and SseJ were found to negatively modulate Sif formation. Our findings demonstrate the complex and dynamic nature of the Sif phenotype.