RESUMEN
Phagocytosis is the process by which myeloid phagocytes bind to and internalize potentially dangerous microorganisms1. During phagocytosis, innate immune receptors and associated signalling proteins are localized to the maturing phagosome compartment, forming an immune information processing hub brimming with microorganism-sensing features2-8. Here we developed proximity labelling of phagosomal contents (PhagoPL) to identify proteins localizing to phagosomes containing model yeast and bacteria. By comparing the protein composition of phagosomes containing evolutionarily and biochemically distinct microorganisms, we unexpectedly identified programmed death-ligand 1 (PD-L1) as a protein that specifically enriches in phagosomes containing yeast. We found that PD-L1 directly binds to yeast upon processing in phagosomes. By surface display library screening, we identified the ribosomal protein Rpl20b as a fungal protein ligand for PD-L1. Using an auxin-inducible depletion system, we found that detection of Rpl20b by macrophages cross-regulates production of distinct cytokines including interleukin-10 (IL-10) induced by the activation of other innate immune receptors. Thus, this study establishes PhagoPL as a useful approach to quantifying the collection of proteins enriched in phagosomes during host-microorganism interactions, exemplified by identifying PD-L1 as a receptor that binds to fungi.
Asunto(s)
Antígeno B7-H1 , Proteínas Fúngicas , Fagosomas , Proteínas Ribosómicas , Saccharomyces cerevisiae , Animales , Femenino , Humanos , Masculino , Ratones , Antígeno B7-H1/metabolismo , Escherichia coli/metabolismo , Proteínas Fúngicas/metabolismo , Interacciones Microbiota-Huesped , Inmunidad Innata , Interleucina-10/metabolismo , Ligandos , Macrófagos/metabolismo , Macrófagos/inmunología , Macrófagos/microbiología , Ratones Endogámicos BALB C , Fagocitosis , Fagosomas/química , Fagosomas/metabolismo , Fagosomas/microbiología , Unión Proteica , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Staphylococcus aureus/metabolismoRESUMEN
Staphylococcus aureus (S. aureus) is one of the most common bacterial infections worldwide, and antibiotic resistant strains such as Methicillin-Resistant S. aureus (MRSA) are a major threat and burden to public health. MRSA not only infects immunocompromised patients but also healthy individuals and has rapidly spread from the healthcare setting to the outside community. However, all vaccines tested in clinical trials to date have failed. Immunocompromised individuals such as patients with HIV or decreased levels of CD4+ T cells are highly susceptible to S. aureus infections, and they are also at increased risk of developing fungal infections. We therefore wondered whether stimulation of antifungal immunity might promote the type of immune responses needed for effective host defense against S. aureus. Here we show that vaccination of mice with a fungal ß-glucan particle (GP) loaded with S. aureus antigens provides protective immunity to S. aureus. We generated glucan particles loaded with the four S. aureus proteins ClfA, IsdA, MntC, and SdrE, creating the 4X-SA-GP vaccine. Vaccination of mice with three doses of 4X-SA-GP promoted protection in a systemic model of S. aureus infection with a significant reduction in the bacterial burden in the spleen and kidneys. 4X-SA-GP vaccination induced antigen-specific Th1 and Th17 CD4+ T cell and antibody responses and provided long-term protection. This work suggests that the GP vaccine system has potential as a novel approach to developing vaccines for S. aureus.
Asunto(s)
Saccharomyces cerevisiae/inmunología , Infecciones Estafilocócicas/inmunología , Vacunas Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Coagulasa/administración & dosificación , Coagulasa/genética , Coagulasa/inmunología , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Saccharomyces cerevisiae/química , Infecciones Estafilocócicas/microbiología , Vacunas Estafilocócicas/administración & dosificación , Vacunas Estafilocócicas/genética , Staphylococcus aureus/genética , Células TH1/inmunología , Células Th17/inmunología , Vacunación , beta-Glucanos/administración & dosificación , beta-Glucanos/inmunologíaRESUMEN
Type I IFNs are a cytokine family essential for antiviral defense. More recently, type I IFNs were shown to be important during bacterial infections. In this article, we show that, in addition to known cytokine functions, IFN-ß is antimicrobial. Parts of the IFN-ß molecular surface (especially helix 4) are cationic and amphipathic, both classic characteristics of antimicrobial peptides, and we observed that IFN-ß can directly kill Staphylococcus aureus Further, a mutant S. aureus that is more sensitive to antimicrobial peptides was killed more efficiently by IFN-ß than was the wild-type S. aureus, and immunoblotting showed that IFN-ß interacts with the bacterial cell surface. To determine whether specific parts of IFN-ß are antimicrobial, we synthesized IFN-ß helix 4 and found that it is sufficient to permeate model prokaryotic membranes using synchrotron x-ray diffraction and that it is sufficient to kill S. aureus These results suggest that, in addition to its well-known signaling activity, IFN-ß may be directly antimicrobial and be part of a growing family of cytokines and chemokines, called kinocidins, that also have antimicrobial properties.
Asunto(s)
Antibacterianos/farmacología , Membrana Celular/efectos de los fármacos , Interferón beta/fisiología , Staphylococcus aureus/efectos de los fármacos , Animales , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Humanos , Interferón beta/química , Interferón beta/metabolismo , Interferón beta/farmacología , Ratones , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Difracción de Rayos XRESUMEN
Innate immune cells must be able to distinguish between direct binding to microbes and detection of components shed from the surface of microbes located at a distance. Dectin-1 (also known as CLEC7A) is a pattern-recognition receptor expressed by myeloid phagocytes (macrophages, dendritic cells and neutrophils) that detects ß-glucans in fungal cell walls and triggers direct cellular antimicrobial activity, including phagocytosis and production of reactive oxygen species (ROS). In contrast to inflammatory responses stimulated upon detection of soluble ligands by other pattern-recognition receptors, such as Toll-like receptors (TLRs), these responses are only useful when a cell comes into direct contact with a microbe and must not be spuriously activated by soluble stimuli. In this study we show that, despite its ability to bind both soluble and particulate ß-glucan polymers, Dectin-1 signalling is only activated by particulate ß-glucans, which cluster the receptor in synapse-like structures from which regulatory tyrosine phosphatases CD45 and CD148 (also known as PTPRC and PTPRJ, respectively) are excluded (Supplementary Fig. 1). The 'phagocytic synapse' now provides a model mechanism by which innate immune receptors can distinguish direct microbial contact from detection of microbes at a distance, thereby initiating direct cellular antimicrobial responses only when they are required.
Asunto(s)
Inmunidad Innata/inmunología , Sinapsis Inmunológicas/inmunología , Proteínas de la Membrana/inmunología , Modelos Inmunológicos , Proteínas del Tejido Nervioso/inmunología , Fagocitosis/inmunología , Animales , Pared Celular/química , Pared Celular/inmunología , Células Cultivadas , Humanos , Lectinas Tipo C , Antígenos Comunes de Leucocito/deficiencia , Antígenos Comunes de Leucocito/metabolismo , Macrófagos/inmunología , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Especies Reactivas de Oxígeno/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/deficiencia , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/inmunología , Transducción de Señal/inmunología , Solubilidad , beta-Glucanos/química , beta-Glucanos/inmunologíaRESUMEN
The importance of type I IFNs in the host response to viral infection is well established; however, their role in bacterial infection is not fully understood. Several bacteria (both Gram-positive and -negative) have been shown to induce IFN-ß production in myeloid cells, but this IFN-ß is not always beneficial to the host. We examined whether Staphylococcus aureus induces IFN-ß from myeloid phagocytes, and if so, whether it is helpful or harmful to the host to do so. We found that S. aureus poorly induces IFN-ß production compared with other bacteria. S. aureus is highly resistant to degradation in the phagosome because it is resistant to lysozyme. Using a mutant that is more sensitive to lysozyme, we show that phagosomal degradation and release of intracellular ligands is essential for induction of IFN-ß and inflammatory chemokines downstream of IFN-ß. Further, we found that adding exogenous IFN-ß during S. aureus infection (in vitro and in vivo) was protective. Together, the data demonstrate that failure to induce IFN-ß production during S. aureus infection contributes to pathogenicity.
Asunto(s)
Interferón beta , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/inmunología , Staphylococcus aureus/patogenicidad , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Interferón beta/antagonistas & inhibidores , Interferón beta/biosíntesis , Interferón beta/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación/inmunología , Infecciones Estafilocócicas/sangre , Staphylococcus aureus/genéticaRESUMEN
The pattern recognition receptors TLR2 and Dectin-1 play key roles in coordinating the responses of macrophages and dendritic cells (DC) to fungi. Induction of proinflammatory cytokines is instructed by signals from both TLR2 and Dectin-1. A recent report identified a role for CARD9 in innate anti-fungal responses, demonstrating CARD9-Bcl10-mediated activation of NF-kappaB and proinflammatory cytokine induction in murine bone marrow-derived DC stimulated via Dectin-1. We now report that Dectin-1-CARD9 signals fail to activate NF-kappaB and drive TNF-alpha induction in murine bone marrow-derived macrophages. However, priming of bone marrow-derived macrophages with GM-CSF or IFN-gamma permits Dectin-1-CARD9-mediated TNF-alpha induction. Analysis of other macrophage/DC populations revealed further variation in the ability of Dectin-1-CARD9 signaling to drive TNF-alpha production. Resident peritoneal cells and alveolar macrophages produce TNF-alpha upon Dectin-1 ligation, while thioglycollate-elicited peritoneal macrophages and Flt3L-derived DC do not. We present data demonstrating that CARD9 is recruited to phagosomes via its CARD domain where it enhances TLR-induced cytokine production even in cells in which Dectin-1 is insufficient to drive cytokine production. In such cells, Dectin-1, CARD9, and Bcl10 levels are not limiting, and data indicate that these cells express additional factors that restrict Dectin-1-CARD9 signaling for TNF-alpha induction.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Proteínas de la Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteína 10 de la LLC-Linfoma de Células B , Células de la Médula Ósea/enzimología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Proteínas Adaptadoras de Señalización CARD , Línea Celular , Células Cultivadas , Células Dendríticas/enzimología , Humanos , Lectinas Tipo C , Macrófagos Alveolares/enzimología , Macrófagos Peritoneales/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Fagosomas/enzimología , Fagosomas/inmunología , Fagosomas/metabolismo , Estructura Terciaria de Proteína , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptores Toll-Like/fisiología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Cryptococcal meningitis is a life-threatening condition most commonly observed in immunocompromised individuals. We describe a daily cannabis smoker without evidence of immunodeficiency presenting with confirmed Cryptococcus neoformans meningitis. An investigation of cannabis samples from the patient's preferred dispensary demonstrated contamination with several varieties of Cryptococcus, including C. neoformans, and other opportunistic fungi. These findings raise concern regarding the safety of dispensary-grade cannabis, even in immunocompetent users.
Asunto(s)
Cryptococcus neoformans , Fumar Marihuana/efectos adversos , Meningitis Criptocócica/microbiología , Femenino , Humanos , Persona de Mediana EdadRESUMEN
The intestinal microflora, typically equated with bacteria, influences diseases such as obesity and inflammatory bowel disease. Here, we show that the mammalian gut contains a rich fungal community that interacts with the immune system through the innate immune receptor Dectin-1. Mice lacking Dectin-1 exhibited increased susceptibility to chemically induced colitis, which was the result of altered responses to indigenous fungi. In humans, we identified a polymorphism in the gene for Dectin-1 (CLEC7A) that is strongly linked to a severe form of ulcerative colitis. Together, our findings reveal a eukaryotic fungal community in the gut (the "mycobiome") that coexists with bacteria and substantially expands the repertoire of organisms interacting with the intestinal immune system to influence health and disease.
Asunto(s)
Colitis Ulcerosa/inmunología , Colitis Ulcerosa/microbiología , Colon/microbiología , Hongos/inmunología , Hongos/fisiología , Mucosa Intestinal/microbiología , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Animales , Anticuerpos Antifúngicos/sangre , Candida tropicalis/inmunología , Candida tropicalis/aislamiento & purificación , Candida tropicalis/patogenicidad , Candida tropicalis/fisiología , Colitis Ulcerosa/inducido químicamente , Colon/inmunología , Recuento de Colonia Microbiana , Sulfato de Dextran , Susceptibilidad a Enfermedades , Femenino , Hongos/clasificación , Hongos/aislamiento & purificación , Haplotipos , Humanos , Inmunidad Innata , Inmunidad Mucosa , Mucosa Intestinal/inmunología , Intestinos/inmunología , Intestinos/microbiología , Lectinas Tipo C/deficiencia , Metagenoma , Ratones , Ratones Endogámicos C57BL , Polimorfismo de Nucleótido SimpleRESUMEN
IL-1beta produced by phagocytes is important for protection against the mucosal pathogen Staphylococcus aureus. Processing and maturation of this cytokine requires activation of the multiprotein inflammasome complex. We observed that the bacterial cell wall component peptidoglycan (PGN) must be particulate and internalized via phagocytosis to activate NLRP3 inflammasomes and IL-1beta secretion. In the context of S. aureus infection of macrophages, we find that phagocytosis and lysozyme-based bacterial cell wall degradation are necessary to induce IL-1beta secretion. Further, an S. aureus enzyme, PGN O-acetyltransferase A, previously demonstrated to make cell wall PGN resistant to lysozyme, strongly suppresses inflammasome activation and inflammation in vitro and in vivo. These observations demonstrate that phagocytosis and lysozyme-based cell wall degradation of S. aureus are functionally coupled to inflammasome activation and IL-1beta secretion and illustrate a case whereby a bacterium specifically subverts IL-1beta secretion through chemical modification of its cell wall PGN.