RESUMEN

Rab5 is required for macropinosome formation, but its site and mode of action remain unknown. We report that Rab5 acts at the plasma membrane, downstream of ruffling, to promote macropinosome sealing and scission. Dominant-negative Rab5, which obliterates macropinocytosis, had no effect on the development of membrane ruffles. However, Rab5-containing vesicles were recruited to circular membrane ruffles, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent endomembrane fusion was necessary for the completion of macropinocytosis. This fusion event coincided with the disappearance of PtdIns(4,5)P2 that accompanies macropinosome closure. Counteracting the depletion of PtdIns(4,5)P2 by expression of phosphatidylinositol-4-phosphate 5-kinase impaired macropinosome formation. Importantly, we found that the removal of PtdIns(4,5)P2 is dependent on Rab5, through the Rab5-mediated recruitment of the inositol 5-phosphatases OCRL and Inpp5b, via APPL1. Knockdown of OCRL and Inpp5b, or APPL1, prevented macropinosome closure without affecting ruffling. We therefore propose that Rab5 is essential for the clearance of PtdIns(4,5)P2 needed to complete the scission of macropinosomes or to prevent their back-fusion with the plasmalemma.

Asunto(s)

Fosfatidilinositol 4,5-Difosfato , Fosfatidilinositoles , Inositol , Inositol Polifosfato 5-Fosfatasas , Pinocitosis

RESUMEN

The neutrophil-specific innate immune receptor CEACAM3 functions as a decoy to capture Gram-negative pathogens, such as Neisseria gonorrhoeae, that exploit CEACAM family members to adhere to the epithelium. Bacterial binding to CEACAM3 results in their efficient engulfment and triggers activation of an nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-dependent inflammatory response by human neutrophils. Herein, we report that CEACAM3 cross-linking is not sufficient for induction of cytokine production and show that the inflammatory response induced by Neisseria gonorrhoeae infection is elicited by an integration of signals from CEACAM3 and toll-like receptors. Using neutrophils from a human CEACAM-expressing mouse line (CEABAC), we use a genetic approach to reveal a molecular bifurcation of the CEACAM3-mediated antimicrobial and inflammatory responses. Ex vivo experiments with CEABAC-Rac2-/- , CEABAC-Bcl10-/- , and CEABAC-Malt1-/- neutrophils indicate that these effectors are not necessary for gonococcal engulfment, yet all 3 effectors contribute to CEACAM3-mediated cytokine production. Interestingly, although Bcl10 and Malt1 are often inextricably linked, Bcl10 enabled synergy between toll-like receptor 4 and CEACAM3, whereas Malt1 did not. Together, these findings reveal an integration of the specific innate immune receptor CEACAM3 into the network of more conventional pattern recognition receptors, providing a mechanism by which the innate immune system can unleash its response to a relentless pathogen.

Asunto(s)

Proteína 10 de la LLC-Linfoma de Células B/genética , Antígeno Carcinoembrionario/inmunología , Gonorrea/inmunología , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genética , Neisseria gonorrhoeae/inmunología , Neutrófilos/inmunología , Receptor Toll-Like 4/inmunología , Animales , Antígenos CD/inmunología , Proteína 10 de la LLC-Linfoma de Células B/inmunología , Moléculas de Adhesión Celular/inmunología , Células Cultivadas , Proteínas Ligadas a GPI/inmunología , Gonorrea/microbiología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/inmunología , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/inmunología , Proteína RCA2 de Unión a GTP

RESUMEN

An overwhelming neutrophil-driven response causes both acute symptoms and the lasting sequelae that result from infection with Neisseria gonorrhoeae. Neutrophils undergo an aggressive opsonin-independent response to N. gonorrhoeae, driven by the innate decoy receptor CEACAM3. CEACAM3 is exclusively expressed by human neutrophils, and drives a potent binding, phagocytic engulfment and oxidative killing of Opa-expressing bacteria. In this study, we sought to explore the contribution of neutrophils to the pathogenic inflammatory process that typifies gonorrhea. Genome-wide microarray and biochemical profiling of gonococcal-infected neutrophils revealed that CEACAM3 engagement triggers a Syk-, PKCδ- and Tak1-dependent signaling cascade that results in the activation of an NF-κB-dependent transcriptional response, with consequent production of pro-inflammatory cytokines. Using an in vivo model of N. gonorrhoeae infection, we show that human CEACAM-expressing neutrophils have heightened migration toward the site of the infection where they may be further activated upon Opa-dependent binding. Together, this study establishes that the role of CEACAM3 is not restricted to the direct opsonin-independent killing by neutrophils, since it also drives the vigorous inflammatory response that typifies gonorrhea. By carrying the potential to mobilize increasing numbers of neutrophils, CEACAM3 thereby represents the tipping point between protective and pathogenic outcomes of N. gonorrhoeae infection.

Asunto(s)

Biomarcadores/metabolismo , Gonorrea/inmunología , Mediadores de Inflamación/metabolismo , Inflamación/etiología , Neisseria gonorrhoeae/patogenicidad , Neutrófilos/inmunología , Animales , Adhesión Bacteriana , Antígeno Carcinoembrionario/genética , Antígeno Carcinoembrionario/metabolismo , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Perfilación de la Expresión Génica , Gonorrea/metabolismo , Gonorrea/microbiología , Humanos , Inflamación/metabolismo , Inflamación/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Transgénicos , Neisseria gonorrhoeae/inmunología , Neutrófilos/microbiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo , Fagocitosis/fisiología , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Quinasa Syk

RESUMEN

Professional phagocytes, such as neutrophils and macrophages, effectively engulf and eliminate invading microorganisms. To survive this onslaught, pathogens have developed an astounding array of countermeasures aimed at avoiding detection, impairing signaling, or paralyzing the machinery that underlies phagocytosis. On the other hand, certain pathogens benefit from attaching to, entering, or traversing host cells to establish and spread infection. This is accomplished by yet other types of effectors that either co-opt or mimic host cell phagocytic components. Here, we briefly summarize the basic features of the phagocytic process and proceed to describe the types of strategies deployed by pathogens to either impair phagocytosis or to gain entry into cells where they can establish a safe survival niche.

Asunto(s)

Bacterias/inmunología , Bacterias/patogenicidad , Interacciones Huésped-Patógeno , Evasión Inmune , Fagocitosis , Animales , Humanos , Macrófagos/inmunología , Modelos Biológicos , Neutrófilos/inmunología

RESUMEN

Some bacteria invade host cells by triggering a process akin to phagocytosis. We analyzed the mechanisms underlying invasion vacuole formation by the bacterium Yersinia pseudotuberculosis. After engaging integrins on host cells, Yersinia resided in PI(4,5)P2-rich, membrane-bound compartments called "prevacuoles" that were inaccessible to extracellular antibodies but remained connected to the plasma membrane. The scission of prevacuoles to form separate, sealed compartments coincided with and required PI(4,5)P2 hydrolysis. At the time of scission, the inositol 5-phosphatases OCRL and Inpp5b were recruited to prevacuoles. This recruitment and subsequent PI(4,5)P2 hydrolysis required the association of the GTPase Rab5 with prevacuoles. Phosphatidylinositol 3-kinase activity was required for fusion of Rab5-positive vesicles with prevacuoles and scission of nascent vacuoles from the plasma membrane. Thus, Yersinia invasion involves a multistep process in which the bacteria form a protective prevacuole and then recruit host factors to induce membrane fission, allowing the bacteria to invade the cell.

Asunto(s)

Membrana Celular/metabolismo , Endocitosis , Fosfatidilinositol 4,5-Difosfato/metabolismo , Yersinia pseudotuberculosis/patogenicidad , Proteínas de Unión al GTP rab5/metabolismo , Animales , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Hidrólisis , Modelos Biológicos , Fosfatidilinositol 3-Quinasa/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Vacuolas/microbiología

RESUMEN

The internalization of foreign or unwanted particles by cells, a process that is important in many aspects of immunity and development, is known as phagocytosis. Rearrangement of cellular actin enables the phagocytic cell to gradually wrap itself around and ultimately engulf the target particle. Phagocytosis is initiated by receptor engagement that in turn triggers multiple signaling pathways, including extensive lipid remodeling. Lipid modification not only generates a variety of second messengers, but is important for the redistribution of key proteins involved in phagocytosis. Lipids can associate with proteins bearing stereospecific association domains. In addition the phosphoinositides and phosphatidylserine (PS), which are anionic, can also recruit proteins electrostatically by generating a considerable negative surface charge. We describe a method whereby lipids can be monitored dynamically in live cells performing phagocytosis. This method involves the expression by phagocytic cells of genetically encoded fluorescent lipid-binding probes, which can be monitored using confocal fluorescence microscopy.

Asunto(s)

Membrana Celular/metabolismo , Fagocitosis , Fosfolípidos/metabolismo , Animales , Sitios de Unión , Técnicas Biosensibles , Células Cultivadas , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Confocal , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de la Célula Individual

RESUMEN

Symptomatic infection of humans with Neisseria gonorrhoeae is characterized by a neutrophil-rich cervical or urethral exudate, suggesting that neutrophils are important both for the clearance of these bacteria and for the pathogenesis of gonorrhea. Neisseria interacts with neutrophils through ligation of human carcinoembryonic antigen related-cellular adhesion molecules (CEACAMs) by their surface-expressed Opa proteins, resulting in bacterial binding, engulfment, and neutrophil activation. Multiple CEACAMs are expressed by human neutrophils, and yet their coexpression has precluded understanding of the relative contribution of each CEACAM to functional responses of neutrophils during neisserial infection. In this work, we directly address the role of each CEACAM during infection by introducing individual human CEACAMs into a differentiated murine MPRO cell line-derived neutrophil model. Murine neutrophils cannot bind the human-restricted Neisseria; however, we show that introducing any of the Opa-binding CEACAMs of human neutrophils (CEACAM1, CEACAM3, and CEACAM6) allows binding and entry of Neisseria into murine neutrophils. While CEACAM1- and CEACAM6-expressing neutrophils bind more bacteria, neisserial uptake via these two receptors unexpectedly proceeds without appreciable neutrophil activation. In stark contrast, neisserial engulfment via CEACAM3 recapitulates the oxidative burst and intracellular granule release seen during human neutrophil infection. Finally, by coexpressing multiple CEACAMs in our model, we show that the expression of CEACAM1 and CEACAM6 potentiate, rather than hinder, CEACAM3-dependent responses of neutrophils, exposing a cooperative role for this family of proteins during neisserial infection of neutrophils. These observations illustrate a divergence in function of CEACAMs in neutrophils and implicate the human-restricted CEACAM3 in the neutrophil innate response to neisserial infection.

Asunto(s)

Antígenos CD/metabolismo , Adhesión Bacteriana , Proteínas de la Membrana Bacteriana Externa/metabolismo , Antígeno Carcinoembrionario/metabolismo , Moléculas de Adhesión Celular/metabolismo , Neisseria gonorrhoeae/patogenicidad , Neutrófilos/microbiología , Animales , Línea Celular , Experimentación Humana , Humanos , Ratones

RESUMEN

Neutrophils are key cells of the innate immune system; they are terminally differentiated and therefore difficult to genetically manipulate and study in vitro. In the present study, we describe a protocol to transiently express two fluorescent markers, the PH domain of protein kinase B fused to red fluorescent protein and the p21-activated kinase-binding domain fused to a yellow fluorescent protein, in primary neutrophils. Using this approach, we are able to achieve a transfection efficiency of approximately 30%. The expression of the transfected probes occurred within 2 h and allowed for real-time monitoring of intermediates in key neutrophil activation pathways at the leading edge of migrating cells. We describe here a transfection protocol for primary neutrophils, which preserves fMLP-mediated cell polarization and cytoskeleton reorganization with simultaneous accumulation of PI-3K products and active Rac at the leading edge. The visualization and analysis of transfected fluorescent markers in primary neutrophils are a powerful technique to monitor chemotaxis signaling pathways in real time.

Asunto(s)

Quimiotaxis de Leucocito , Proteínas Luminiscentes/metabolismo , Neutrófilos/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Membrana Celular/metabolismo , Proteínas Luminiscentes/genética , Ratones , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/genética , Receptores de Formil Péptido/genética , Receptores de Formil Péptido/metabolismo , Quinasas p21 Activadas , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismo , Proteína Fluorescente Roja

RESUMEN

The human-restricted pathogens Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae and Moraxella catarrhalis colonize host tissues via carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs). One such receptor, CEACAM3, acts in a host-protective manner by orchestrating the capture and engulfment of invasive bacteria by human neutrophils. Herein, we show that bacterial binding to CEACAM3 causes recruitment of the cytoplasmic tyrosine kinase Syk, resulting in the phosphorylation of both CEACAM3 and Syk. This interaction is specific for the immunoreceptor tyrosine-based activation motif (ITAM) in the CEACAM3 cytoplasmic domain. While dispensable for the phagocytic uptake of single bacteria by CEACAM3, Syk is necessary for internalization when cargo size increases or when the density of CEACAM-binding ligand on the cargo surface is below a critical threshold. Moreover, Syk engagement is required for an effective bacterial killing response, including the neutrophil oxidative burst and degranulation functions in response to N. gonorrhoeae. These data reveal CEACAM3 as a specific innate immune receptor that mediates the opsonin-independent clearance of CEACAM-binding bacteria via Syk, a molecular trigger for functional immunoreceptor responses of both the adaptive (TCR, BCR, FcR) and innate (Dectin-1, CEACAM3) immune systems.

Asunto(s)

Antígeno Carcinoembrionario/inmunología , Inmunidad Innata/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neutrófilos/inmunología , Proteínas Tirosina Quinasas/metabolismo , Gonorrea/metabolismo , Células HeLa , Humanos , Neisseria gonorrhoeae/inmunología , Neisseria gonorrhoeae/patogenicidad , Neutrófilos/microbiología , Estructura Terciaria de Proteína , Transducción de Señal/fisiología , Quinasa Syk

RESUMEN

Human adenoviruses are common pathogens associated with many diseases, including respiratory, gastrointestinal, and ocular infections. Because they are now being increasingly recognized as agents of life-threatening disseminated infection in immunocompromised patients, robust and sensitive laboratory detection methods are needed for their rapid diagnosis. We describe here a PCR assay using a single primer pair, targeting a region of the hexon gene containing hypervariable region 7, that can detect all known human adenovirus serotypes and allows for serotype determination through the analysis of the nucleotide sequence. This comprehensive assay has proven effective for diagnosing adenoviruses at the serotype level in a broad range of patient specimens, including conjunctival, nasopharyngeal, stool, blood, and urine specimens.

Asunto(s)

Adenovirus Humanos/clasificación , Adenovirus Humanos/genética , Adenovirus Humanos/aislamiento & purificación , Secuencia de Bases , Cartilla de ADN , Humanos , Filogenia , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y Especificidad