RESUMO
Ingestion and killing of bacteria by phagocytic cells protect the human body against infections. While many mechanisms have been proposed to account for bacterial killing in phagosomes, their relative importance, redundancy, and specificity remain unclear. In this study, we used the Dictyostelium discoideum amoeba as a model phagocyte and quantified the requirement of 11 individual gene products, including nine putative effectors, for the killing of bacteria. This analysis revealed that radically different mechanisms are required to kill Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis AlyL, a lysozyme-like protein equipped with a distinct bacteriolytic region, plays a specific role in the intracellular killing of K. pneumoniae, with assistance from BpiC and Aoah, two lipopolysaccharide (LPS)-binding proteins. Rapid killing of E. coli and P. aeruginosa requires the presence of BpiC and of the NoxA NADPH oxidase. No single effector tested is essential for rapid killing of S. aureus or B. subtilis Overall, our observations reveal an unsuspected degree of specificity in the elimination of bacteria in phagosomes.IMPORTANCE Phagocytic cells ingest and kill bacteria, a process essential for the defense of the human body against infections. Many potential killing mechanisms have been identified in phagocytic cells, including free radicals, toxic ions, enzymes, and permeabilizing peptides. Yet fundamental questions remain unanswered: what is the relative importance of these mechanisms, how redundant are they, and are different mechanisms used to kill different species of bacteria? We addressed these questions using Dictyostelium discoideum, a model phagocytic cell amenable to genetic manipulations and quantitative analysis. Our results reveal that vastly different mechanisms are required to kill different species of bacteria. This very high degree of specificity was unexpected and indicates that a lot remains to be discovered about how phagocytic cells eliminate bacteria.
Assuntos
Bactérias/imunologia , Dictyostelium/genética , Dictyostelium/microbiologia , Fagócitos/microbiologia , Bactérias/classificação , Dictyostelium/imunologia , Klebsiella pneumoniae/imunologia , Fagócitos/imunologia , Fagocitose , Fagossomos , Pseudomonas aeruginosa/imunologia , Staphylococcus aureus/imunologiaRESUMO
Engulfment of extracellular material by phagocytosis or macropinocytosis depends on the ability of cells to generate specialized cup-shaped protrusions. To effectively capture and internalize their targets, these cups are organized into a ring or ruffle of actin-driven protrusion encircling a non-protrusive interior domain. These functional domains depend on the combined activities of multiple Ras and Rho family small GTPases, but how their activities are integrated and differentially regulated over space and time is unknown. Here, we show that the amoeba Dictyostelium discoideum coordinates Ras and Rac activity using the multidomain protein RGBARG (RCC1, RhoGEF, BAR, and RasGAP-containing protein). We find RGBARG uses a tripartite mechanism of Ras, Rac, and phospholipid interactions to localize at the protruding edge and interface with the interior of both macropinocytic and phagocytic cups. There, we propose RGBARG shapes the protrusion by expanding Rac activation at the rim while suppressing expansion of the active Ras interior domain. Consequently, cells lacking RGBARG form enlarged, flat interior domains unable to generate large macropinosomes. During phagocytosis, we find that disruption of RGBARG causes a geometry-specific defect in engulfing rod-shaped bacteria and ellipsoidal beads. This demonstrates the importance of coordinating small GTPase activities during engulfment of more complex shapes and thus the full physiological range of microbes, and how this is achieved in a model professional phagocyte.
Assuntos
Bactérias , Dictyostelium/citologia , Dictyostelium/metabolismo , Dictyostelium/fisiologia , Fagocitose , Pinocitose , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Proteínas de Ciclo Celular , Dictyostelium/imunologiaRESUMO
The social amoeba Dictyostelium discoideum is a tractable model organism to study cellular allorecognition, which is the ability of a cell to distinguish itself and its genetically similar relatives from more distantly related organisms. Cellular allorecognition is ubiquitous across the tree of life and affects many biological processes. Depending on the biological context, these versatile systems operate both within and between individual organisms, and both promote and constrain functional heterogeneity. Some of the most notable allorecognition systems mediate neural self-avoidance in flies and adaptive immunity in vertebrates. D. discoideum's allorecognition system shares several structures and functions with other allorecognition systems. Structurally, its key regulators reside at a single genomic locus that encodes two highly polymorphic proteins, a transmembrane ligand called TgrC1 and its receptor TgrB1. These proteins exhibit isoform-specific, heterophilic binding across cells. Functionally, this interaction determines the extent to which co-developing D. discoideum strains co-aggregate or segregate during the aggregation phase of multicellular development. The allorecognition system thus affects both development and social evolution, as available evidence suggests that the threat of developmental cheating represents a primary selective force acting on it. Other significant characteristics that may inform the study of allorecognition in general include that D. discoideum's allorecognition system is a continuous and inclusive trait, it is pleiotropic, and it is temporally regulated.
Assuntos
Dictyostelium/imunologia , Dictyostelium/fisiologia , Imunidade Adaptativa , Adesão Celular , Quimiotaxia , Dictyostelium/genética , Glicoproteínas/genética , Glicosilação , Ligantes , Modelos Biológicos , Fenótipo , Proteínas de Protozoários/genéticaRESUMO
Integrated with both a historical perspective and an evolutionary angle, this opinion article presents a brief and personal view of the emergence of cellular microbiology research. From the very first observations of phagocytosis by Goeze in 1777 to the exhaustive analysis of the cellular defence mechanisms performed in modern laboratories, the studies by cell biologists and microbiologists have converged into an integrative research field distinct from, but fully coupled to immunity: cellular microbiology. In addition, this brief article is thought as a humble patchwork of the motivations that have guided the research in my group over a quarter century.
Assuntos
Dictyostelium/imunologia , Mycobacterium marinum/imunologia , Fagocitose/imunologia , Animais , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/microbiologia , Dictyostelium/ultraestrutura , História do Século XVIII , História do Século XIX , História do Século XXI , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Microbiologia/história , Mycobacterium marinum/crescimento & desenvolvimento , Mycobacterium marinum/patogenicidade , Fagossomos/imunologia , Fagossomos/microbiologia , Fagossomos/ultraestruturaRESUMO
The professional phagocyte Dictyostelium discoideum is a well-established model organism to study host-pathogen interactions. Dictyostelium amoebae grow as separate, independent cells; they divide by binary fission and take up bacteria and yeast via phagocytosis. In the year 2000, D. discoideum was described by two groups as a novel system for genetic analysis of host-pathogen interactions for the intracellular pathogen Legionella pneumophila. Since then additional microbial pathogens that can be studied in D. discoideum have been reported. The organism has various advantages for the dissection of the complex cross-talk between a host and a pathogen. A fully sequenced and well-curated genome is available, there are excellent molecular genetic tools on the market, and the generation of targeted multiple gene knock-outs as well as the realization of untargeted genetic screens is generally straightforward. Dictyostelium also offers easy cultivation, and the cells are suitable for cell biological studies, which in combination with in vivo expression of fluorescence-tagged proteins allows the investigation of the dynamics of bacterial uptake and infection. Furthermore, a large mutant collection is available at the Dictyostelium stock center, favoring the identification of host resistance or susceptibility genes. Here, we briefly describe strategies to identify host cell factors important during an infection, followed by protocols for cell culture and storage, uptake and infection, and confocal microscopy of infected cells.
Assuntos
Dictyostelium/microbiologia , Interações Hospedeiro-Patógeno , Legionella/fisiologia , Legionelose/microbiologia , Amoeba/microbiologia , Dictyostelium/genética , Dictyostelium/imunologia , Citometria de Fluxo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Microscopia Confocal , Mutação , FagocitoseRESUMO
Microbes have interacted with eukaryotic cells for as long as they have been co-existing. While many of these interactions are beneficial for both the microbe as well as the eukaryotic cell, several microbes have evolved into pathogenic species. For some of these pathogens, host cell invasion results in irreparable damage and thus host cell destruction, whereas others use the host to avoid immune detection and elimination. One of the latter pathogens is Mycobacterium tuberculosis, arguably one of the most notorious pathogens on earth. In mammalian macrophages, M. tuberculosis manages to survive within infected macrophages by avoiding intracellular degradation in lysosomes using a number of different strategies. One of these is based on the recruitment and phagosomal retention of the host protein coronin 1, that is a member of the coronin protein family and a mammalian homolog of coronin A, a protein identified in Dictyostelium. Besides mediating mycobacterial survival in macrophages, coronin 1 is also an important regulator of naïve T cell homeostasis. How, exactly, coronin 1 mediates its activity in immune cells remains unclear. While in lower eukaryotes coronins are involved in cytoskeletal regulation, the functions of the seven coronin members in mammals are less clear. Dictyostelium coronins may have maintained multiple functions, whereas the mammalian coronins may have evolved from regulators of the cytoskeleton to modulators of signal transduction. In this minireview, we will discuss the different studies that have contributed to understand the molecular and cellular functions of coronin proteins in mammals and Dictyostelium.
Assuntos
Dictyostelium/imunologia , Mamíferos/imunologia , Proteínas dos Microfilamentos/imunologia , Fagócitos/imunologia , Tuberculose/imunologia , Animais , Dictyostelium/genética , Humanos , Mamíferos/genética , Mamíferos/microbiologia , Proteínas dos Microfilamentos/genética , Mycobacterium tuberculosis/fisiologia , Tuberculose/genética , Tuberculose/microbiologiaRESUMO
Due to their archaic life style and microbivor behavior, amoebae may represent a source of antimicrobial peptides and proteins. The amoebic protozoon Dictyostelium discoideum has been a model organism in cell biology for decades and has recently also been used for research on host-pathogen interactions and the evolution of innate immunity. In the genome of D. discoideum, genes can be identified that potentially allow the synthesis of a variety of antimicrobial proteins. However, at the protein level only very few antimicrobial proteins have been characterized that may interact directly with bacteria and help in fighting infection of D. discoideum with potential pathogens. Here, we focus on a large group of gene products that structurally belong to the saposin-like protein (SAPLIP) family and which members we named provisionally Apls (amoebapore-like peptides) according to their similarity to a comprehensively studied antimicrobial and cytotoxic pore-forming protein of the protozoan parasite Entamoeba histolytica. We focused on AplD because it is the only Apl gene that is reported to be primarily transcribed further during the multicellular stages such as the mobile slug stage. Upon knock-out (KO) of the gene, aplD- slugs became highly vulnerable to virulent Klebsiella pneumoniae. AplD- slugs harbored bacterial clumps in their interior and were unable to slough off the pathogen in their slime sheath. Re-expression of AplD in aplD- slugs rescued the susceptibility toward K. pneumoniae. The purified recombinant protein rAplD formed pores in liposomes and was also capable of permeabilizing the membrane of live Bacillus megaterium. We propose that the multifarious Apl family of D. discoideum comprises antimicrobial effector polypeptides that are instrumental to interact with bacteria and their phospholipid membranes. The variety of its members would allow a complementary and synergistic action against a variety of microbes, which the amoeba encounters in its environment.
Assuntos
Infecções Bacterianas/imunologia , Dictyostelium/imunologia , Dictyostelium/microbiologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Saposinas/metabolismo , Saposinas/farmacologia , Animais , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Bacillus megaterium/efeitos dos fármacos , Dictyostelium/genética , Dictyostelium/metabolismo , Gastrópodes/imunologia , Gastrópodes/metabolismo , Gastrópodes/microbiologia , Perfilação da Expressão Gênica , Canais Iônicos/metabolismo , Canais Iônicos/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/patogenicidade , Lipossomos/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Peptídeos/farmacologia , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/farmacologia , Proteínas Recombinantes , Saposinas/genética , Saposinas/imunologiaRESUMO
The Francisella genus comprises highly pathogenic bacteria that can cause fatal disease in their vertebrate and invertebrate hosts including humans. In general, Francisella growth depends on iron availability, hence, iron homeostasis must be tightly regulated during Francisella infection. We used the system of the professional phagocyte Dictyostelium and the fish pathogen F. noatunensis subsp. noatunensis (F.n.n.) to investigate the role of the host cell iron transporters Nramp (natural resistance associated macrophage proteins) during Francisella infection. Like its mammalian ortholog, Dictyostelium Nramp1 transports iron from the phagosome into the cytosol, whereas the paralog NrampB is located on the contractile vacuole and controls, together with Nramp1, the cellular iron homeostasis. In Dictyostelium, Nramp1 localized to the F.n.n.-phagosome but disappeared from the compartment dependent on the presence of IglC, an established Francisella virulence factor. In the absence of Nramp transporters the bacteria translocated more efficiently from the phagosome into the host cell cytosol, its replicative niche. Increased escape rates coincided with increased proteolytic activity in bead-containing phagosomes indicating a role of the Nramp transporters for phagosomal maturation. In the nramp mutants, a higher bacterial load was observed in the replicative phase compared to wild-type host cells. Upon bacterial access to the cytosol of wt cells, mRNA levels of bacterial iron uptake factors were transiently upregulated. Decreased iron levels in the nramp mutants were compensated by a prolonged upregulation of the iron scavenging system. These results show that Nramps contribute to host cell immunity against Francisella infection by influencing the translocation efficiency from the phagosome to the cytosol but not by restricting access to nutritional iron in the cytosol.
Assuntos
Proteínas de Transporte de Cátions/farmacologia , Dictyostelium/imunologia , Dictyostelium/microbiologia , Francisella/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/veterinária , Interações Hospedeiro-Patógeno/imunologia , Ferro/metabolismo , Animais , Carga Bacteriana/efeitos dos fármacos , Proteínas de Transporte de Cátions/genética , Citosol/metabolismo , Citosol/microbiologia , Dictyostelium/metabolismo , Peixes/imunologia , Peixes/microbiologia , Francisella/genética , Francisella/metabolismo , Francisella/patogenicidade , Técnicas de Inativação de Genes , Infecções por Bactérias Gram-Negativas/microbiologia , Homeostase , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Transporte de Íons/fisiologia , Fagocitose , Fagossomos/microbiologia , Fagossomos/fisiologia , Fatores de Virulência/metabolismoRESUMO
The impact of cellular individuality on host-microbe interactions is increasingly appreciated but studying the temporal dynamics of single-cell behavior in this context remains technically challenging. Here we present a microfluidic platform, InfectChip, to trap motile infected cells for high-resolution time-lapse microscopy. This approach allows the direct visualization of all stages of infection, from bacterial uptake to death of the bacterium or host cell, over extended periods of time. We demonstrate the utility of this approach by co-culturing an established host-cell model, Dictyostelium discoideum, with the extracellular pathogen Klebsiella pneumoniae or the intracellular pathogen Mycobacterium marinum. We show that the outcome of such infections is surprisingly heterogeneous, ranging from abortive infection to death of the bacterium or host cell. InfectChip thus provides a simple method to dissect the time-course of host-microbe interactions at the single-cell level, yielding new insights that could not be gleaned from conventional population-based measurements.
Assuntos
Rastreamento de Células/instrumentação , Técnicas de Cocultura/instrumentação , Interações Hospedeiro-Patógeno , Dispositivos Lab-On-A-Chip , Modelos Biológicos , Fagocitose , Análise de Célula Única/instrumentação , Animais , Células Cultivadas , Células Imobilizadas , Desenho Assistido por Computador , Dictyostelium/citologia , Dictyostelium/imunologia , Dictyostelium/fisiologia , Dictyostelium/ultraestrutura , Dimetilpolisiloxanos/química , Desenho de Equipamento , Humanos , Interpretação de Imagem Assistida por Computador , Klebsiella pneumoniae/citologia , Klebsiella pneumoniae/imunologia , Klebsiella pneumoniae/fisiologia , Klebsiella pneumoniae/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica de Varredura , Mycobacterium marinum/citologia , Mycobacterium marinum/imunologia , Mycobacterium marinum/fisiologia , Mycobacterium marinum/ultraestrutura , Fagócitos/citologia , Fagócitos/imunologia , Fagócitos/microbiologia , Fagócitos/parasitologia , Imagem com Lapso de TempoRESUMO
Dictyostelium discoideum has been used largely as a model organism to study the organization and function of the endocytic pathway. Here we describe dense structures present in D. discoideum endocytic compartments, which we named pycnosomes. Pycnosomes are constitutively secreted in the extracellular medium, from which they can be recovered by differential centrifugation. We identified the most abundant protein present in secreted pycnosomes, that we designated SctA. SctA defines a new family of proteins with four members in D. discoideum, and homologous proteins in other protists and eumetazoa. We developed a monoclonal antibody specific for SctA and used it to further characterize secreted and intracellular pycnosomes. Within cells, immunofluorescence as well as electron microscopy identified pycnosomes as SctA-enriched dense structures in the lumen of endocytic compartments. Pycnosomes are occasionally seen in continuity with intra-endosomal membranes, particularly in U18666A-treated cells where intraluminal budding is highly enhanced. While the exact nature, origin and cellular function of pycnosomes remain to be established, this study provides a first description of these structures as well as a characterization of reagents that can be used for further studies.
Assuntos
Dictyostelium/metabolismo , Endossomos/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Transporte Biológico , Dictyostelium/imunologia , Dictyostelium/ultraestrutura , Endocitose , Endossomos/imunologia , Endossomos/ultraestrutura , Membranas Intracelulares/metabolismo , Camundongos , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismoRESUMO
Many bacteria can resist phagocytic digestion by various protozoa. Some of these bacteria (all human pathogens) are known to be packaged in multilamellar bodies produced in the phagocytic pathway of the protozoa and that are secreted into the extracellular milieu. Packaged bacteria are protected from harsh conditions, and the packaging process is suspected to promote bacterial persistence in the environment. To date, only a limited number of protozoa, belonging to free-living amoebae and ciliates, have been shown to perform bacteria packaging. It is still unknown if social amoebae can do bacteria packaging. The link between the capacity of 136 bacterial isolates to resist the grazing of the social amoeba Dictyostelium discoideum and to be packaged by this amoeba was investigated in the present study. The 45 bacterial isolates displaying a resisting phenotype were tested for their capacity to be packaged. A total of seven isolates from Cupriavidus, Micrococcus, Microbacterium and Rathayibacter genera seemed to be packaged and secreted by D. discoideum based on immunofluorescence results. Electron microscopy confirmed that the Cupriavidus and Rathayibacter isolates were formally packaged. These results show that social amoebae can package some bacteria from the environment revealing a new aspect of microbial ecology.
Assuntos
Bactérias/isolamento & purificação , Dictyostelium/microbiologia , Bactérias/classificação , Bactérias/genética , Dictyostelium/imunologia , Dados de Sequência Molecular , FilogeniaRESUMO
The evolutionarily conserved catabolic process of autophagy involves the degradation of cytoplasmic components through lysosomal enzymes. Basal levels of autophagy maintain cellular homeostasis and under stress conditions high levels of autophagy are induced. It is often under such stress conditions that high levels of autophagy and cell death have been observed, leading to the idea that autophagy may act as an executioner of cell death. However the notion of autophagy as a cell death mechanism has been controversial and remains mechanistically undefined. There is now growing evidence that in specific contexts autophagy can indeed facilitate cell death. The pro-death role of autophagy is however complicated due to the extensive cross-talk between different signalling pathways. This review summarises the examples of where autophagy acts as a means of cell death and discusses the association of autophagy with the different cell death pathways.
Assuntos
Apoptose/genética , Autofagia/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Fagossomos/genética , Enzimas de Conjugação de Ubiquitina/genética , Animais , Apoptose/imunologia , Autofagia/imunologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/imunologia , Dictyostelium/genética , Dictyostelium/imunologia , Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Lisossomos/genética , Lisossomos/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Fagossomos/imunologia , Transdução de Sinais , Estresse Fisiológico , Enzimas de Conjugação de Ubiquitina/imunologiaRESUMO
BACKGROUND: Immunolocalization of cellular antigens typically requires fixation and permeabilization of cells, prior to incubation with antibodies. FINDINGS: Assessing a test protein abundantly present at the cell surface of Dictyostelium cells, we show that in fixed cells, permeabilization extracts almost completely this cell surface antigen. The extent of this artifact is variable depending on the procedure used for labeling and permeabilization, as well as on the antigen considered. CONCLUSIONS: An optimized protocol for labeling both surface and intracellular antigens without significant loss of labeling is proposed.
Assuntos
Antígenos de Superfície/análise , Dictyostelium/imunologia , Imunofluorescência/métodos , Antígenos de Superfície/imunologiaRESUMO
The loading controls used for quantitative immunoblotting of mammalian proteins are not appropriate for use with Dictyostelium discoideum. Actin levels, for example, change greatly during Dictyostelium development. In addition, Dictyostelium-specific antibodies for other potential control proteins are not commercially available. Here we demonstrate the use of labeled streptavidin to detect biotinylated mitochondrial 3-methylcrotonyl-CoA carboxylase α (MCCC1), providing a robust and convenient tool for quantitative normalization of Dictyostelium Western blots, as well as fluorescently labeling mitochondria for microscopy of fixed cells.
Assuntos
Dictyostelium/química , Immunoblotting/normas , Mitocôndrias/metabolismo , Proteínas Mitocondriais/análise , Estreptavidina/química , Biomarcadores/análise , Carbono-Carbono Ligases/análise , Carbono-Carbono Ligases/química , Carbono-Carbono Ligases/metabolismo , Dictyostelium/imunologia , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Mitocôndrias/química , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismoRESUMO
The Bacillus cereus group bacteria contain pathogens of economic and medical importance. From security and health perspectives, the lethal mammalian pathogen Bacillus anthracis remains a serious threat. In addition the potent insect pathogen Bacillus thuringiensis is extensively used as a biological control agent for insect pests. This relies upon the industrial scale induction of bacterial spore formation with the associated production of orally toxic Cry-toxins. Understanding the ecology and potential alternative developmental fates of these bacteria is therefore important. Here we describe the use of an amoeba host model to investigate the influence of environmental bactivorous protists on both spores and vegetative cells of these pathogens. We demonstrate that the bacteria can respond to different densities of amoeba by adopting different behaviours and developmental fates. We show that spores will germinate in response to factors excreted by the amoeba, and that the bacteria can grow and reproduce on these factors. We show that in low densities of amoeba, that the bacteria will seek to colonise the surface of the amoeba as micro-colonies, resisting phagocytosis. At high amoeba densities, the bacteria change morphology into long filaments and macroscopic rope-like structures which cannot be ingested due to size exclusion. We suggest these developmental fates are likely to be important both in the ecology of these bacteria and also during animal host colonisation and immune evasion.
Assuntos
Acanthamoeba/imunologia , Bacillus anthracis/crescimento & desenvolvimento , Bacillus cereus/crescimento & desenvolvimento , Bacillus thuringiensis/crescimento & desenvolvimento , Dictyostelium/imunologia , Acanthamoeba/fisiologia , Bacillus anthracis/fisiologia , Bacillus cereus/fisiologia , Bacillus thuringiensis/fisiologia , Quimiotaxia , Dictyostelium/fisiologia , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Fagocitose , Especificidade da Espécie , Esporos Bacterianos/crescimento & desenvolvimentoRESUMO
Innate immune cells respond to microbial invaders using pattern recognition receptors that detect conserved microbial patterns. Among the cellular processes stimulated downstream of pattern recognition machinery is the initiation of autophagy, which plays protective roles against intracellular microbes. We have shown recently that Dictyostelium discoideum, which takes up bacteria for nutritive purposes, may employ pattern recognition machinery to respond to bacterial prey, as D. discoideum cells upregulate bactericidal activity upon stimulation by lipopolysaccharide (LPS). Here we extend these findings, showing that LPS treatment leads to induction of autophagosomal maturation in cells responding to the bacteria Staphylococcus aureus. Cells treated with the autophagy-inducing drug rapamycin clear internalized bacteria at an accelerated rate, while LPS-enhanced clearance of bacteria is reduced in cells deficient for the autophagy-related genes atg1 and atg9. These findings link microbial pattern recognition with autophagy in the social amoeba D. discoideum.
Assuntos
Autofagia/imunologia , Dictyostelium/microbiologia , Lipopolissacarídeos/imunologia , Fagossomos/microbiologia , Antibacterianos/farmacologia , Autofagia/efeitos dos fármacos , Dictyostelium/imunologia , Fagossomos/imunologia , Sirolimo/farmacologia , Staphylococcus aureus/imunologiaRESUMO
Phagocytosis resistance is an important virulence factor in Klebsiella pneumoniae. Dictyostelium has been used to study the interaction between phagocytes and bacteria because of its similarity to mammalian macrophages. In this study, we used a Dictyostelium model to investigate genes for resistance to phagocytosis in NTUH-K2044, a strain of K. pneumoniae causing pyogenic liver abscess that is highly resistant to phagocytosis. A total of 2,500 transposon mutants were screened by plaque assay, and 29 of them permitted phagocytosis by Dictyostelium. In the 29 mutants, six loci were identified; three were capsular synthesis genes. Of the other three, one was related to carnitine metabolism, one encoded a subunit of protease (clpX), and one encoded a lipopolysaccharide O-antigen transporter (wzm). Deletion and complementation of these genes showed that only ΔclpX and Δwzm mutants became susceptible to Dictyostelium phagocytosis, and their complementation restored the phagocytosis resistance phenotype. These two mutants were also susceptible to phagocytosis by human neutrophils and revealed attenuated virulence in a mouse model, implying that they play important roles in the pathogenesis of K. pneumoniae. Furthermore, we demonstrated that clpP, which exists in an operon with clpX, was also involved in resistance to phagocytosis. The transcriptional profile of ΔclpX was examined by microarray analysis and revealed a 3-fold lower level of expression of capsular synthesis genes. Therefore, we have identified genes involved in resistance to phagocytosis in K. pneumoniae using Dictyostelium, and this model is useful to explore genes associated with resistance to phagocytosis in heavily encapsulated bacteria.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/genética , Dictyostelium/imunologia , Endopeptidase Clp/genética , Klebsiella pneumoniae/genética , Fagocitose , Transportadores de Cassetes de Ligação de ATP/imunologia , Animais , Proteínas de Bactérias/imunologia , Elementos de DNA Transponíveis , Endopeptidase Clp/imunologia , Feminino , Loci Gênicos , Humanos , Immunoblotting , Klebsiella pneumoniae/imunologia , Klebsiella pneumoniae/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Análise em Microsséries , Microscopia Confocal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , VirulênciaRESUMO
Infection of Dictyostelium discoideum with Legionella pneumophila resulted in a large number of differentially regulated genes among them three core autophagy genes, ATG8, ATG9 and ATG16. Macroautophagy contributes to many physiological and pathological processes and might also constitute an important mechanism in cell-autonomous immunity. For further studies we selected the highly conserved ATG9. In colocalization studies with GFP-tagged ATG9 and different organelle marker proteins we neither observed colocalization with mitochondria, the ER nor lysosomes. However, there was partial colocalization with the Golgi apparatus and many ATG9-GFP-containing vesicles localized along microtubules and accumulated around the microtubule organizing centre. ATG9-deficient cells had pleiotropic defects. In addition to growth defects they displayed severe developmental defects, consistent with the known role of autophagy in Dictyostelium development. Unexpectedly, the ATG9 mutant also had a strong phagocytosis defect that was particularly apparent when infecting the cells with L. pneumophila. However, those Legionellae that entered the host could multiply better in mutant than in wild-type cells, because of a less efficient clearance in the early and a more efficient replication in the late phase of infection. We conclude that ATG9 and hence macroautophagy has a protective role during pathogen infection.
Assuntos
Dictyostelium/genética , Legionella pneumophila/crescimento & desenvolvimento , Fagocitose , Proteínas de Protozoários/genética , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/imunologia , Dictyostelium/microbiologia , Técnicas de Inativação de Genes , Complexo de Golgi/química , Microtúbulos/química , Proteínas de Protozoários/análise , Proteínas de Protozoários/fisiologiaRESUMO
Antibodies were produced against fragments of the microtubule-binding domain and the motor domain of the dynein heavy chain from Dictyostelium discoideum to probe whole cell extracts of root meristem cells of wheat Triticum aestivum. In plant extracts, these antibodies cross-reacted with a polypeptide of high molecular weight (>500kDa). The antibodies bound to protein A-Sepharose precipitated high molecular weight polypeptide from cell extracts. Immunofluorescence showed that the antibodies identified various aggregates inside cells, localized at the perinuclear area during interphase to early prophase, at the spindle periphery and polar area during mitosis, and in the interzonal region during phragmoplast development. Some aggregates were also co-labeled by markers for the Golgi apparatus. Thus, we found in higher plant cells a high molecular weight antigen cross-reacting with the antibodies to motor and microtubule-binding domains of dynein heavy chains. This antigen is associated with aggregates distributed in the cytoplasm in cell cycle-dependent manner. A subset of these aggregates belongs to the Golgi complex.
Assuntos
Anticorpos Antiprotozoários/imunologia , Dineínas/imunologia , Complexo de Golgi/química , Proteínas de Plantas/imunologia , Sequência de Aminoácidos , Animais , Reações Cruzadas , Dictyostelium/imunologia , Complexo de Golgi/imunologia , Interfase , Dados de Sequência Molecular , Peptídeos/análise , Peptídeos/imunologia , Proteínas de Plantas/análise , Prófase , Triticum/imunologiaRESUMO
Human leukocytes, including macrophages and neutrophils, are phagocytic immune cells that capture and engulf pathogens and subsequently destroy them in intracellular vesicles. To accomplish this vital task, these leukocytes utilize two basic cell behaviors-chemotaxis for chasing down infectious pathogens and phagocytosis for destroying them. The molecular mechanisms controlling these behaviors are not well understood for immune cells. Interestingly, a soil amoeba, Dictyostelium discoideum, uses these same behaviors to pursue and injest its bacterial food source and to organize its multi-cellular development. Consequently, studies of this model system have provided and will continue to provide us with mechanistic insights into the chemotaxis and phagocytosis of immune cells. Here, we review recent research in these areas that have been conducted in the Chemotaxis Signal Section of NIAID's Laboratory of Immunogenetics.