RESUMEN
Bacteria, including those that are pathogenic, have been generally classified according to their ability to survive and grow in the presence or absence of oxygen: aerobic and anaerobic bacteria, respectively. Strict aerobes require oxygen to grow (e.g., Neisseria), and strict anaerobes grow exclusively without, and do not survive oxygen exposure (e.g., Clostridia); aerotolerant bacteria (e.g., Lactobacilli) are insensitive to oxygen exposure. Facultative anaerobes (e.g., E. coli) have the unique ability to grow in the presence or in the absence of oxygen and are thus well-adapted to these changing conditions, which may constitute an underestimated selective advantage for infection. In the WHO antibiotic-resistant 'priority pathogens' list, facultative anaerobes are overrepresented (8 among 12 listed pathogens), consistent with clinical studies performed in populations particularly susceptible to infectious diseases. Bacteria aerobic respiratory chain plays a central role in oxygen consumption, leading to the formation of hypoxic infectious sites (infectious hypoxia). Facultative anaerobes have developed a wide diversity of aerotolerance and anaerotolerance strategies in vivo. However, at a single cell level, the modulation of the intracellular oxygen level in host infected cells remains elusive and will be discussed in this review. In conclusion, the ability of facultative bacteria to evolve in the presence or the absence of oxygen is essential for their virulence strategy and constitute a selective advantage. TAKE AWAY: Most life-threatening pathogenic bacteria are facultative anaerobes. Only facultative anaerobes are aerotolerant, anaerotolerant and capable of consuming O2 . Facultative anaerobes induce and are well adapted to cellular hypoxia.
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Escherichia coli , Oxígeno , Bacterias , Bacterias Anaerobias , Consumo de OxígenoRESUMEN
Neutrophils are the most abundant circulating white blood cells and are the central players of the innate immune response. During their lifecycle, neutrophils mainly evolve under low oxygen conditions (0.1-4% O2 ), to which they are well adapted. Neutrophils are atypical cells since they are highly glycolytic and susceptible to oxygen exposure, which induces their activation and death through mechanisms that remain currently elusive. Nevertheless, nearly all studies conducted on neutrophils are carried out under atmospheric oxygen (21%), corresponding to hyperoxia. Here, we investigated the impact of hyperoxia during neutrophil purification and culture on neutrophil viability, activation and cytosolic protein content. We demonstrate that neutrophil hyper-activation (CD62L shedding) is induced during culture under hyperoxic conditions (24 h), compared with neutrophils cultured under anoxic conditions. Spontaneous neutrophil extracellular trap (NET) formation is observed when neutrophils face hyperoxia during purification or culture. In addition, we show that maintaining neutrophils in autologous plasma is the preferred strategy to maintain their basal state. Our results show that manipulating neutrophils under hyperoxic conditions leads to the loss of 57 cytosolic proteins during purification, while it does not lead to an immediate impact on neutrophil activation (CD11bhigh , CD54high , CD62Lneg ) or viability (DAPI+ ). We identified two clusters of proteins belonging to cholesterol metabolism and to the complement and coagulation cascade pathways, which are highly susceptible to neutrophil oxygen exposure during neutrophil purification. In conclusion, protecting neutrophil from oxygen during their purification and culture is recommended to avoid activation and to prevent the alteration of cytosolic protein composition.
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Trampas Extracelulares , Neutrófilos , Recuento de Leucocitos , Activación Neutrófila , OxígenoRESUMEN
Polymorphonuclear neutrophils (PMNs) are innate immune cells, which represent 50% to 70% of the total circulating leukocytes. How PMNs adapt to various microenvironments encountered during their life cycle, from the bone marrow, to the blood plasma fraction, and to inflamed or infected tissues remains largely unexplored. Metabolic shifts have been reported in other immune cells such as macrophages or lymphocytes, in response to local changes in their microenvironment, and in association with a modulation of their pro-inflammatory or anti-inflammatory functions. The potential contribution of metabolic shifts in the modulation of neutrophil activation or survival is anticipated even though it is not yet fully described. If neutrophils are considered to be mainly glycolytic, the relative importance of alternative metabolic pathways, such as the pentose phosphate pathway, glutaminolysis, or the mitochondrial oxidative metabolism, has not been fully considered during activation. This statement may be explained by the lack of knowledge regarding the local availability of key metabolites such as glucose, glutamine, and substrates, such as oxygen from the bone marrow to inflamed tissues. As highlighted in this review, the link between specific metabolic pathways and neutrophil activation has been outlined in many reports. However, the impact of neutrophil activation on metabolic shifts' induction has not yet been explored. Beyond its importance in neutrophil survival capacity in response to available metabolites, metabolic shifts may also contribute to neutrophil population heterogeneity reported in cancer (tumor-associated neutrophil) or auto-immune diseases (Low/High Density Neutrophils). This represents an active field of research. In conclusion, the characterization of neutrophil metabolic shifts is an emerging field that may provide important knowledge on neutrophil physiology and activation modulation. The related question of microenvironmental changes occurring during inflammation, to which neutrophils will respond to, will have to be addressed to fully appreciate the importance of neutrophil metabolic shifts in inflammatory diseases.
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Redes y Vías Metabólicas/inmunología , Mitocondrias/inmunología , Activación Neutrófila , Neutrófilos/inmunología , Animales , Supervivencia Celular/inmunología , Humanos , Inflamación/inmunología , Inflamación/patología , Mitocondrias/patología , Neutrófilos/patología , Oxidación-ReducciónRESUMEN
Functional studies of human neutrophils and their transfusion for clinical purposes have been hampered by their short life span after isolation. Here, we demonstrate that neutrophil viability is maintained for 20 hours in culture media at 37°C under anoxic conditions with 3 mM glucose and 32 µg/mL dimethyloxalylglycine supplementation, as evidenced by stabilization of Mcl-1, proliferating cell nuclear antigen (PCNA), and pro-caspase-3. Notably, neutrophil morphology (nucleus shape and cell-surface markers) and functions (phagocytosis, degranulation, calcium release, chemotaxis, and reactive oxygen species production) were comparable to blood circulating neutrophils. The observed extension in neutrophil viability was reversed upon exposure to oxygen. Extending neutrophil life span allowed efficient transfection of plasmids (40% transfection efficiency) and short interfering RNA (interleukin-8, PCNA, and Bax), as a validation of effective and functional genetic manipulation of neutrophils both in vitro and in vivo. In vivo, transfusion of conditioned neutrophils in a neutropenic guinea pig model increased bacterial clearance of Shigella flexneri upon colonic infection, strongly suggesting that these conditioned neutrophils might be suitable for transfusion purposes. In summary, such conditioning of neutrophils in vitro should facilitate their study and offer new opportunities for genetic manipulation and therapeutic use.
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Glucosa/farmacología , Hipoxia/patología , Neutrófilos/citología , Animales , Antiinfecciosos/metabolismo , Apoptosis/efectos de los fármacos , Biomarcadores/metabolismo , Transfusión Sanguínea , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Separación Celular , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cobayas , Humanos , Neutrófilos/efectos de los fármacos , Neutrófilos/ultraestructura , Oxígeno/farmacología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Transfección , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Imaging living cells and organs requires innovative, specific, efficient, and well tolerated fluorescent markers targeting cellular components. Such tools will allow proceeding to the dynamic analysis of cells and the adaptation of tissues to environmental cues. In this study, we have identified and synthesized a novel non-toxic fluorescent marker allowing a specific fluorescent staining of the human colonic mucus. Our strategy to identify a molecule able to specifically bind to the human colonic mucus was on the basis of the mucus adhesion properties of commensal bacteria. We identified and characterized the mucus-binding property of a 70-amino acid domain (MUB(70)) expressed on the surface of Lactobacillus strains. The chemical synthesis of MUB(70) was achieved using the human commensal bacterium Lactobacillus reuteri AF120104 protein as a template. The synthesized Cy5-conjugated MUB(70) marker specifically stained the colonic mucus on fixed human, rabbit, and guinea pig tissues. Interestingly, murine tissue was not stained, suggesting significant differences in the composition of the murine colonic mucus. In addition, this marker stained the mucus of living cultured human colonic cells (HT29-MTX) and human colonic tissue explants. Using a biotinylated derivative of MUB(70), we demonstrated that this peptide binds specifically to Muc2, the most abundant secreted mucin, through its glycosylated moieties. Hence, Cy5-MUB(70) is a novel and specific fluorescent marker for mammalian colonic mucus. It may be used for live imaging analysis but also, as demonstrated in this study, as a marker for the diagnosis and the prognosis of colonic mucinous carcinomas.
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Proteínas Bacterianas/metabolismo , Colon/metabolismo , Limosilactobacillus reuteri/metabolismo , Mucina 2/metabolismo , Moco/metabolismo , Secuencia de Aminoácidos , Animales , Adhesión Bacteriana , Proteínas Bacterianas/química , Proteínas Bacterianas/farmacología , Supervivencia Celular , Colon/microbiología , Electroforesis en Gel de Poliacrilamida , Colorantes Fluorescentes/química , Glicosilación , Cobayas , Células HT29 , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Inmunohistoquímica , Limosilactobacillus reuteri/genética , Limosilactobacillus reuteri/fisiología , Ratones , Microscopía Fluorescente , Datos de Secuencia Molecular , Moco/microbiología , Unión Proteica , Conejos , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
BACKGROUND: Shigella sp. are enteric pathogens which causes >125 million cases of shigellosis annually. S. sonnei accounts for about a quarter of those cases and is increasingly prevalent in industrialising nations. Being an enteric pathogen, S. sonnei benefits from outcompeting gut commensals such as Escherichia coli to establish itself and cause disease. There are numerous mechanisms that bacterial pathogens use to outcompete its rivals including molecules called colicins. A Type 6 Secretion System (T6SS) was recently described as contributing to E. coli killing in S. sonnei. METHODS: We used Bulk Phenotyping of Epidemiological Replicates (BPER) which combined bacterial Genome Wide Association Studies (bGWAS) and high throughput phenotyping on a collection of S. sonnei surveillance isolates to identify the genetic features associated with E. coli killing and explore their relationship with epidemiological behaviour. We further explored the presence of colicins and T6SS components in the isolates using genomics, laboratory experimentation, and proteomics. FINDINGS: Our bGWAS analysis returned known and novel colicin and colicin related genes as significantly associated with E. coli killing. In silico analyses identified key colicin clusters responsible for the killing phenotype associated with epidemiologically successful sub-lineages. The killing phenotype was not associated with the presence of a T6SS. Laboratory analyses confirmed the presence of the key colicin clusters and that killing was contact-independent. INTERPRETATION: Colicins are responsible for E. coli killing by S. sonnei, not a T6SS. This phenotype contributes to shaping the observed epidemiology of S. sonnei and may contribute to its increasing prevalence globally. BPER is an epidemiologically relevant approach to phenotypic testing that enables the rapid identification of genetic drivers of phenotypic changes, and assessment of their relevance to epidemiology in natural settings. FUNDING: Biotechnology and Biological Sciences Research Council, Biotechnology and Biological Sciences Research Council Doctoral Training Partnership studentship, Wellcome Trust, Medical Research Council (UK), French National Research Agency.
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Colicinas , Shigella , Humanos , Colicinas/genética , Escherichia coli/genética , Shigella sonnei/genética , Estudio de Asociación del Genoma CompletoRESUMEN
Shigella spp. are the causative agents of bacterial dysentery and shigellosis, mainly in children living in developing countries. The study of Shigella entire life cycle in vivo and the evaluation of vaccine candidates' protective efficacy have been hampered by the lack of a suitable animal model of infection. None of the studies evaluated so far (rabbit, guinea pig, mouse) allowed the recapitulation of full shigellosis symptoms upon Shigella oral challenge. Historical reports have suggested that dysentery and scurvy are both metabolic diseases associated with ascorbate deficiency. Mammals, which are susceptible to Shigella infection (humans, non-human primates and guinea pigs) are among the few species unable to synthesize ascorbate. We optimized a low-ascorbate diet to induce moderate ascorbate deficiency, but not scurvy, in guinea pigs to investigate whether poor vitamin C status increases the progression of shigellosis. Moderate ascorbate deficiency increased shigellosis symptom severity during an extended period of time (up to 48 h) in all strains tested (Shigella sonnei, Shigella flexneri 5a, and 2a). At late time points, an important influx of neutrophils was observed both within the disrupted colonic mucosa and in the luminal compartment, although Shigella was able to disseminate deep into the organ to reach the sub-mucosal layer and the bloodstream. Moreover, we found that ascorbate deficiency also increased Shigella penetration into the colon epithelium layer in a Gulo-/- mouse infection model. The use of these new rodent models of shigellosis opens new doors for the study of both Shigella infection strategies and immune responses to Shigella infection.
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Disentería Bacilar , Microbioma Gastrointestinal , Shigella , Cobayas , Humanos , Animales , Conejos , Ratones , Disentería Bacilar/microbiología , Modelos Animales de Enfermedad , Shigella flexneri , Ácido Ascórbico , MamíferosRESUMEN
Bacterial and fungal pathogens face various microenvironmental conditions during infection. In addition to acidosis, nutrient consumption, and hypercapnia, pathogen infections are associated with hypoxia, which is induced by bacterial and fungal respiration during the formation of foci of infection or biofilms. Consequently, the in vivo interaction between host immune cells and pathogens is anticipated to occur mainly under low-oxygen conditions. Various infectious disease models have reported that pathogens benefit from hypoxia, which dampens the oxygen-dependent antimicrobial activities of macrophages and neutrophils, such as the production of reactive oxygen species (ROS). Due to their dual respiration capacity (aerobic and anaerobic) or phenotypical adaptation (e.g., dormancy), pathogens have the capacity to survive and disseminate in the absence of oxygen. In addition, hypoxia modulates various mechanisms of pathogen virulence, promoting the dissemination of pathogens. Further investigations are still required to evaluate the relative importance of oxygen on the capacity of pathogens to invade and colonize host organs and to better understand alternative strategies developed by immune cells to circumvent pathogen dissemination in the absence of oxygen. Addressing this important and fundamental question in various models of infection may direct the development of innovative therapeutic strategies.
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Micosis , Oxígeno , Humanos , Hipoxia , Neutrófilos , Especies Reactivas de Oxígeno , VirulenciaRESUMEN
In human blood, oxygen is mainly transported by red blood cells. Accordingly, the dissolved oxygen level in plasma is expected to be limited, although it has not been quantified yet. Here, by developing dedicated methods and tools, we determined that human plasma pO2 = 8.4 mmHg (1.1% O2). Oxygen solubility in plasma was believed to be similar to water. Here we reveal that plasma has an additional ascorbate-dependent oxygen-reduction activity. Plasma experimental oxygenation oxidizes ascorbate (49.5 µM in fresh plasma vs < 2 µM in oxidized plasma) and abolishes this capacity, which is restored by ascorbate supplementation. We confirmed these results in vivo, showing that the plasma pO2 is significantly higher in ascorbate-deficient guinea pigs (Ascorbateplasma < 2 µM), compared to control (Ascorbateplasma > 15 µM). Plasma low oxygen level preserves the integrity of oxidation-sensitive components such as ubiquinol. Circulating leucocytes are well adapted to these conditions, since the abundance of their mitochondrial network is limited. These results shed a new light on the importance of oxygen exposure on leucocyte biological study, in regards with the reducing conditions they encounter in vivo; but also, on the manipulation of blood products to improve their integrity and potentially improve transfusions' efficacy.
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Ácido Ascórbico/sangre , Ácido Ascórbico/metabolismo , Oxígeno/sangre , Plasma/metabolismo , Animales , Línea Celular , Linaje de la Célula/fisiología , Eritrocitos/metabolismo , Cobayas , Células HEK293 , Células Hep G2 , Humanos , Hipoxia/sangre , Hipoxia/metabolismo , Oxidación-Reducción , Solubilidad , Ubiquinona/análogos & derivados , Ubiquinona/metabolismoRESUMEN
Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high concentrations in specific and tertiary granules of neutrophils. This protocol details how MUB40 conjugated directly to a fluorophore can be used to stain neutrophils in fixed/permeabilized tissues as well as how this can be used in live-cell imaging to assay for neutrophil activation and de-granulation. Neutrophil detection methods are limited to species-specific monoclonal antibodies, which are not always suitable for certain applications. MUB40 does not penetrate the cell membrane and is thus excluded from lactoferrin stored in non-activated/non-permeabilized neutrophils. MUB40 has the added benefit of recognizing lactoferrin from a broad host range, making it especially useful for comparing results in studies involving multiple research models, reducing the number of duplicate reagents, and simplifying protocols through single-step staining.
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Mediadores de Inflamación/metabolismo , Neutrófilos/inmunología , Péptidos/metabolismo , HumanosRESUMEN
Shigella spp. are bacterial pathogens that invade the human colonic mucosa using a type III secretion apparatus (T3SA), a proteinaceous device activated upon contact with host cells. Active T3SAs translocate proteins that carve the intracellular niche of Shigella spp. Nevertheless, the activation state of the T3SA has not been addressed in vivo. Here, we used a green fluorescent protein transcription-based secretion activity reporter (TSAR) to provide a spatio-temporal description of S. flexneri T3SAs activity in the colon of Guinea pigs. First, we observed that early mucus release is triggered in the vicinity of luminal bacteria with inactive T3SA. Subsequent mucosal invasion showed bacteria with active T3SA associated with the brush border, eventually penetrating into epithelial cells. From 2 to 8 h post-challenge, the infection foci expanded, and these intracellular bacteria displayed homogeneously high-secreting activity, while extracellular foci within the lamina propria featured bacteria with low secretion activity. We also found evidence that within lamina propria macrophages, bacteria reside in vacuoles instead of accessing the cytosol. Finally, bacteria were cleared from tissues between 8 and 24 h post-challenge, highlighting the hit-and-run colonization strategy of Shigella. This study demonstrates how genetically encoded reporters can contribute to deciphering pathogenesis in vivo.
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Colon/microbiología , Disentería Bacilar/microbiología , Shigella flexneri/fisiología , Sistemas de Secreción Tipo III/fisiología , Animales , Biomarcadores , Modelos Animales de Enfermedad , Femenino , Genes Reporteros , Cobayas , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Especificidad de Órganos , Distribución TisularRESUMEN
Pathogenic enterobacteria face various oxygen (O2) levels during intestinal colonization from the O2-deprived lumen to oxygenated tissues. Using Shigella flexneri as a model, we have previously demonstrated that epithelium invasion is promoted by O2 in a type III secretion system-dependent manner. However, subsequent pathogen adaptation to tissue oxygenation modulation remained unknown. Assessing single-cell distribution, together with tissue oxygenation, we demonstrate here that the colonic mucosa O2 is actively depleted by S. flexneri aerobic respiration-and not host neutrophils-during infection, leading to the formation of hypoxic foci of infection. This process is promoted by type III secretion system inactivation in infected tissues, favouring colonizers over explorers. We identify the molecular mechanisms supporting infectious hypoxia induction, and demonstrate here how enteropathogens optimize their colonization capacity in relation to their ability to manipulate tissue oxygenation during infection.
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Disentería Bacilar/metabolismo , Mucosa Intestinal/microbiología , Oxígeno/metabolismo , Shigella flexneri/patogenicidad , Animales , Hipoxia de la Célula , Modelos Animales de Enfermedad , Disentería Bacilar/microbiología , Femenino , Cobayas , Células Hep G2 , Humanos , Mucosa Intestinal/metabolismo , Conejos , Shigella flexneri/metabolismo , Sistemas de Secreción Tipo III/metabolismoRESUMEN
Neutrophil granules (NGs) are key components of the innate immune response and mark the development of neutrophilic granulocytes in mammals. However, there has been no specific fluorescent vital stain up to now to monitor their dynamics within a whole live organism. We rationally designed a benzochalcone fluorescent probe (HAB) featuring high tissue permeability and optimal photophysics such as elevated quantum yield, pronounced solvatochromism and target-induced fluorogenesis. Phenotypic screening identified HAB as the first cell- and organelle-specific small-molecule fluorescent tracer of NGs in live zebrafish larvae, with no labeling of other cell types or organelles. HAB staining was independent of the state of neutrophil activation, labeling NGs of both resting and phagocytically active neutrophils with equal specificity. By high-resolution live imaging, we documented the dynamics of HAB-stained NGs during phagocytosis. Upon zymosan injection, labeled NGs were rapidly recruited to the forming phagosomes. Despite being a reversible ligand, HAB could not be displaced by high concentrations of pharmacologically relevant competing chalcones, indicating that this specific labeling was the result of the HAB's precise physicochemical signature rather than a general feature of chalcones. However, one of the competitors was discovered as a promising interstitial fluorescent tracer illuminating zebrafish histology, similarly to BODIPY-ceramide. As a yellow-emitting histopermeable vital stain, HAB functionally and spectrally complements most genetically incorporated fluorescent tags commonly used in live zebrafish biology, holding promise for the study of neutrophil-dependent responses relevant to human physiopathology such as developmental defects, inflammation and infection. Furthermore, HAB intensely labeled isolated live human neutrophils at the level of granulated subcellular structures consistent with human NGs, suggesting that the labeling of NGs by HAB is not restricted to the zebrafish model but also relevant to mammalian systems.
RESUMEN
Neutrophils represent the most abundant immune cells recruited to inflamed tissues. A lack of dedicated tools has hampered their detection and study. We show that a synthesized peptide, MUB40, binds to lactoferrin, the most abundant protein stored in neutrophil-specific and tertiary granules. Lactoferrin is specifically produced by neutrophils among other leukocytes, making MUB40 a specific neutrophil marker. Naive mammalian neutrophils (human, guinea pig, mouse, rabbit) were labeled by fluorescent MUB40 conjugates (-Cy5, Dylight405). A peptidase-resistant retro-inverso MUB40 (RI-MUB40) was synthesized and its lactoferrin-binding property validated. Neutrophil lactoferrin secretion during in vitro Shigella infection was assessed with RI-MUB40-Cy5 using live cell microscopy. Systemically administered RI-MUB40-Cy5 accumulated at sites of inflammation in a mouse arthritis inflammation model in vivo and showed usefulness as a potential tool for inflammation detection using non-invasive imaging. Improving neutrophil detection with the universal and specific MUB40 marker will aid the study of broad ranges of inflammatory diseases.
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Carbocianinas/química , Colorantes Fluorescentes/química , Inflamación/diagnóstico , Lactoferrina/análisis , Neutrófilos/inmunología , Péptidos/química , Adulto , Animales , Biomarcadores/análisis , Disentería Bacilar/complicaciones , Disentería Bacilar/diagnóstico , Disentería Bacilar/inmunología , Disentería Bacilar/microbiología , Femenino , Cobayas , Humanos , Inflamación/complicaciones , Inflamación/inmunología , Inflamación/microbiología , Lactoferrina/inmunología , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Neutrófilos/microbiología , Conejos , Shigella/inmunologíaRESUMEN
More than two decades after cloning the cystic fibrosis transmembrane regulator (CFTR) gene, the defective gene in cystic fibrosis (CF), we still do not understand how dysfunction of this ion channel causes lung disease and the tremendous neutrophil burden which persists within the airways; nor why chronic colonization by Pseudomonas aeruginosa develops in CF patients who are thought to be immunocompetent. It appears that the microenvironment within the lung of CF patients provides favorable conditions for both P. aeruginosa colonization and neutrophil survival. In this context, the ability of bacteria to induce hypoxia, which in turn affects neutrophil survival is an additional level of complexity that needs to be accounted for when controlling neutrophil fate in CF. Recent studies have underscored the importance of neutrophils in innate immunity and their functions appear to extend far beyond their well-described role in antibacterial defense. Perhaps a disturbance in neutrophil reprogramming during the course of an infection severely modulates the inflammatory response in CF. Furthermore there is an emerging concept that the CFTR itself may be an immune modulator and stimulating CFTR function in CF patients could promote neutrophil and macrophages antimicrobial function. Fostering the resolution of inflammation by favoring neutrophil apoptosis could preserve their microbicidal activities but decrease their proinflammatory potential. In this context, triggering neutrophil apoptosis with roscovitine may be a potential therapeutic option and this is currently being evaluated in CF patients. In the present review we discuss how neutrophils functions are disturbed in CF and how this may relate to chronic infection with P. aeuginosa and we propose novel research directions aimed at modulating neutrophil survival, dampening lung inflammation and ultimately leading to an amelioration of the lung disease.
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Fibrosis Quística/inmunología , Infecciones/inmunología , Inflamación/inmunología , Neutrófilos/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/patogenicidad , Animales , Antibacterianos/farmacología , Apoptosis , Fibrosis Quística/complicaciones , Fibrosis Quística/microbiología , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/inmunología , Humanos , Hipoxia , Inmunidad Innata , Pulmón/microbiología , Pulmón/patología , Enfermedades Pulmonares/etiología , Enfermedades Pulmonares/microbiología , Macrófagos/inmunología , Ratones , Neutrófilos/microbiología , Infecciones por Pseudomonas/etiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/inmunología , Purinas/farmacología , Roscovitina , VirulenciaRESUMEN
Hypoxia is defined as a tissue oxygenation status below physiological needs. During Shigella infection, an infectious hypoxia is induced within foci of infection. In this review, we discuss how Shigella physiology and virulence are modulated and how the main recruited immune cells, the neutrophils, adapt to this environment.
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Disentería Bacilar/microbiología , Disentería Bacilar/patología , Hipoxia/patología , Inmunidad Innata , Neutrófilos/inmunología , Shigella/inmunología , Shigella/patogenicidad , Animales , Modelos Animales de Enfermedad , HumanosRESUMEN
Shigella is a leading cause of dysentery worldwide, with the majority of infections caused by two subgroups, S. flexneri and S. sonnei. Although S. flexneri has been highly prevalent in low-income countries, global development has brought an increase in S. sonnei at the expense of S. flexneri. However, the mechanisms behind this shift are not understood. Here we report that S. sonnei, but not S. flexneri, encodes a type VI secretion system (T6SS) that provides a competitive advantage in the gut. S. sonnei competes against E. coli and S. flexneri in mixed cultures, but this advantage is reduced in T6SS mutant strains. In addition, S. sonnei can persist as well as outcompete E. coli and S. flexneri in mice in a T6SS-dependent manner. These findings suggest that S. sonnei has a competitive advantage over S. flexneri and potentially explain the increasing global prevalence of S. sonnei.
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Disentería Bacilar/microbiología , Shigella sonnei/metabolismo , Sistemas de Secreción Tipo VI/genética , Sistemas de Secreción Tipo VI/fisiología , Animales , Antibiosis/fisiología , Técnicas de Cocultivo , Colon/microbiología , Colon/patología , Recuento de Colonia Microbiana , Modelos Animales de Enfermedad , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Femenino , Cobayas , Lactobacillus/crecimiento & desarrollo , Ratones , Ratones Endogámicos BALB C , Interacciones Microbianas , Mutación , Shigella flexneri/efectos de los fármacos , Shigella flexneri/genética , Shigella flexneri/crecimiento & desarrollo , Shigella sonnei/genética , Shigella sonnei/crecimiento & desarrollo , Sistemas de Secreción Tipo VI/farmacologíaRESUMEN
Somatic gain-of-function mutations in isocitrate dehydrogenases (IDH) 1 and 2 are found in multiple hematologic and solid tumors, leading to accumulation of the oncometabolite (R)-2-hydroxyglutarate (2HG). 2HG competitively inhibits α-ketoglutarate-dependent dioxygenases, including histone demethylases and methylcytosine dioxygenases of the TET family, causing epigenetic dysregulation and a block in cellular differentiation. In vitro studies have provided proof of concept for mutant IDH inhibition as a therapeutic approach. We report the discovery and characterization of AG-221, an orally available, selective, potent inhibitor of the mutant IDH2 enzyme. AG-221 suppressed 2HG production and induced cellular differentiation in primary human IDH2 mutation-positive acute myeloid leukemia (AML) cells ex vivo and in xenograft mouse models. AG-221 also provided a statistically significant survival benefit in an aggressive IDH2R140Q-mutant AML xenograft mouse model. These findings supported initiation of the ongoing clinical trials of AG-221 in patients with IDH2 mutation-positive advanced hematologic malignancies.Significance: Mutations in IDH1/2 are identified in approximately 20% of patients with AML and contribute to leukemia via a block in hematopoietic cell differentiation. We have shown that the targeted inhibitor AG-221 suppresses the mutant IDH2 enzyme in multiple preclinical models and induces differentiation of malignant blasts, supporting its clinical development. Cancer Discov; 7(5); 478-93. ©2017 AACR.See related commentary by Thomas and Majeti, p. 459See related article by Shih et al., p. 494This article is highlighted in the In This Issue feature, p. 443.
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Aminopiridinas/farmacología , Antineoplásicos/farmacología , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Leucemia Mieloide Aguda/genética , Triazinas/farmacología , Animales , Línea Celular Tumoral , Humanos , Isocitrato Deshidrogenasa/genética , Ratones , Mutación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Shigella is a pathovar of Escherichia coli comprising four groups, Shigella flexneri, Shigella sonnei, Shigella dysenteriae, and Shigella boydii, each of them, with the exception of S.sonnei, comprising several serotypes. Shigella accounts for the majority of dysentery causing infections occurring world-wide each year. Recent advancements in the Shigella field have led to a better understanding of the molecular mechanisms underlying host epithelial cell invasion and immune cell function manipulation, mainly using S. flexneri as a model. Host-cell invasion is the final step of the infection process, as Shigella's virulence strategy relies also on its ability to survive hostile conditions during its journey through the gastro-intestinal tract, to compete with the host microbiota and to cross the intestinal mucus layer. Hence, the diversity of the virulence strategies among the different Shigella species has not yet been deeply investigated, which might be an important step to understand the epidemiological spreading of Shigella species worldwide and a key aspect for the validation of novel vaccine candidates. The recent development of high-throughput screening and sequencing methods will facilitate these complex comparison studies. In this review we discuss several of the major avenues that the Shigella research field has taken over the past few years and hopefully gain some insights into the questions that remain surrounding this important human pathogen.
Asunto(s)
Disentería Bacilar/epidemiología , Shigella boydii/patogenicidad , Shigella dysenteriae/patogenicidad , Shigella flexneri/patogenicidad , Shigella sonnei/patogenicidad , Disentería Bacilar/inmunología , Disentería Bacilar/microbiología , Células Epiteliales/microbiología , Geografía , Interacciones Huésped-Patógeno , HumanosRESUMEN
Bacterial cell division requires the formation of a mature divisome complex positioned at the midcell. The localization of the divisome complex is determined by the correct positioning, assembly, and constriction of the FtsZ ring (Z-ring). Z-ring constriction control remains poorly understood and (to some extent) controversial, probably due to the fact that this phenomenon is transient and controlled by numerous factors. Here, we characterize ZapE, a novel ATPase found in Gram-negative bacteria, which is required for growth under conditions of low oxygen, while loss of zapE results in temperature-dependent elongation of cell shape. We found that ZapE is recruited to the Z-ring during late stages of the cell division process and correlates with constriction of the Z-ring. Overexpression or inactivation of zapE leads to elongation of Escherichia coli and affects the dynamics of the Z-ring during division. In vitro, ZapE destabilizes FtsZ polymers in an ATP-dependent manner. IMPORTANCE Bacterial cell division has mainly been characterized in vitro. In this report, we could identify ZapE as a novel cell division protein which is not essential in vitro but is required during an infectious process. The bacterial cell division process relies on the assembly, positioning, and constriction of FtsZ ring (the so-called Z-ring). Among nonessential cell division proteins recently identified, ZapE is the first in which detection at the Z-ring correlates with its constriction. We demonstrate that ZapE abundance has to be tightly regulated to allow cell division to occur; absence or overexpression of ZapE leads to bacterial filamentation. As zapE is not essential, we speculate that additional Z-ring destabilizing proteins transiently recruited during late cell division process might be identified in the future.