Asunto(s)
Ectima/inmunología , Enfermedades de Inmunodeficiencia Primaria/diagnóstico , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/aislamiento & purificación , Sepsis/inmunología , Piel/patología , Ectima/diagnóstico , Ectima/patología , Resultado Fatal , Gangrena , Humanos , Lactante , Quinasas Asociadas a Receptores de Interleucina-1 , Masculino , Enfermedades de Inmunodeficiencia Primaria/complicaciones , Infecciones por Pseudomonas/diagnóstico , Infecciones por Pseudomonas/patología , Sepsis/diagnóstico , Sepsis/patología , Piel/inmunología , Piel/microbiologíaRESUMEN
Pathogens phagocytosis and the uptake of apoptotic cells (efferocytosis) are essential macrophages tasks, classically considered as mutually exclusive. Macrophages have been observed to polarize into either pro-inflammatory/microbicidal or anti-inflammatory/efferocytic phenotypes. However, macrophage functions have shown to be more complex. Furthermore, little is known about the regulation of efferocytosis under inflammatory conditions. In this study, we elucidate the modulation of the macrophage efferocytic function during an inflammatory stimulus. We find that bone marrow-derived macrophages (BMDM) are very efficient in engulfing both the bacterial pathogen Pseudomonas aeruginosa and apoptotic cells. BMDM showed a high bactericidal capacity unaffected by the concomitant presence of apoptotic material. Plasticity in macrophage programming, in response to changing environmental cues, may modulate efferocytic capability. In this work, we further show that, after phagocyting and processing Pseudomonas aeruginosa, macrophages highly increase their efferocytic capacity without affecting their phagocytic function. Moreover, we demonstrate that Pseudomonas aeruginosa enhances efferocytosis of these phagocytes through the IL-6 signaling pathway. Our results show that the inflammatory response generated by the bacterial processing enhances these macrophages' capacity to control inflammation through an increased efferocytosis.
Asunto(s)
Apoptosis , Macrófagos/inmunología , Macrófagos/microbiología , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/fisiología , Células Cultivadas , Citocinas/metabolismo , Mediadores de Inflamación/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Fagocitosis , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/patologíaRESUMEN
Pseudomonas aeruginosa is considered an opportunistic pathogen of great clinical importance. The clearance of this bacterium occurs through recognition of the pathogen by innate immune system receptors, leading to a lung inflammatory response. However, this response must be controlled via immunoregulatory pathways. In this study, we evaluate the role of endogenous murine IL-10 after acute infection with the virulent strain P. aeruginosa PA14. To assess the role of IL-10, intratracheal infection with the PA14 strain was performed in C57BL/6 or IL-10 KO mice. The PA14 strain was recovered in both types of animals, although IL-10 KO mice presented a higher number of viable bacteria in the lung when compared to the C57BL/6 group. Histopathological and stereological analyses showed that IL-10 KO mice had higher tissue damage and inflammatory infiltrate when compared to control animals. The activity of MMP-9 but not MMP-2, as well as IL-6 and TNF-α expression, were augmented in the lungs of infected animals and was much more evident in IL-10 KO animals when compared to the other analyzed groups. This work indicates that endogenous IL-10 control P. aeruginosa infection, the expression of pro-inflammatory genes, MMP-9 activity and histopathological processes of the infectious process in question.
Asunto(s)
Interleucina-10/inmunología , Pulmón , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Animales , Inmunidad , Pulmón/inmunología , Pulmón/patología , Metaloproteinasa 9 de la Matriz/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Pseudomonas aeruginosa (PA) is an opportunistic human pathogen responsible for causing serious infections in patients with cystic fibrosis. Infections caused by PA are difficult to treat and eradicate due to intrinsic and added resistance to antibiotic therapy. Therefore, it is necessary to establish effective prevention strategies against this infectious agent. In this study, a combination of immunoinformatic tools was applied to predict immunogenic and immunodominant regions in the structure of exotoxins commonly secreted as virulence factors in PA infection (ExoA, ExoS, ExoT, ExoU and ExoY). The peptides derived from exotoxins were evaluated for the potential affinity for human leukocyte antigen (HLA) I and HLA-II molecules, antigenicity score and toxicity profile. From an initial screening of 941 peptides, 13 (1.38%) were successful in all analyzes. The peptides with relevant immunogenic properties were mainly those derived from Exo A (10 / 76.9%). All peptides selected in the last analysis present a high population coverage rate based on the interaction of HLA alleles (95.36 ± 7.83%). Therefore, the peptides characterized in this study are recommended for in vitro and in vivo studies and can provide the basis for the rational design of a vaccine against PA.
Asunto(s)
Exotoxinas/química , Exotoxinas/inmunología , Antígenos HLA/química , Péptidos/química , Péptidos/inmunología , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/inmunología , Secuencia de Aminoácidos , Toxinas Bacterianas/inmunología , Simulación por Computador , Epítopos/química , Epítopos/inmunología , Antígenos HLA/inmunología , Humanos , Inmunogenicidad Vacunal , Simulación del Acoplamiento Molecular , Conformación Proteica , Infecciones por Pseudomonas/inmunología , Vacunas/química , Vacunas/inmunología , Factores de Virulencia/química , Factores de Virulencia/inmunologíaRESUMEN
Pseudomonas aeruginosa is one of the most common opportunistic pathogens causing respiratory infections in hospitals. Vancomycin, the antimicrobial agent usually used to treat bacterial nosocomial infections, is associated with gut dysbiosis. As a lung-gut immunologic axis has been described, this study aimed to evaluate both the immunologic and histopathologic effects on the lungs and the large intestine resulting from vancomycin-induced gut dysbiosis in the P. aeruginosa pneumonia murine model. Metagenomic analysis demonstrated that vancomycin-induced gut dysbiosis resulted in higher Proteobacteria and lower Bacteroidetes populations in feces. Given that gut dysbiosis could augment the proinflammatory status of the intestines leading to a variety of acute inflammatory diseases, bone marrow-derived macrophages were stimulated with cecal content from dysbiotic mice showing a higher expression of proinflammatory cytokines and lower expression of IL-10. Dysbiotic mice showed higher levels of viable bacteria in the lungs and spleen when acutely infected with P. aeruginosa, with more lung and cecal damage and increased IL-10 expression in bronchoalveolar lavage. The susceptible and tissue damage phenotype was reversed when dysbiotic mice received fecal microbiota transplantation. In spite of higher recruitment of CD11b+ cells in the lungs, there was no higher CD80+ expression, DC+ cell amounts or proinflammatory cytokine expression. Taken together, our results indicate that the bacterial community found in vancomycin-induced dysbiosis dysregulates the gut inflammatory status, influencing the lung-gut immunologic axis to favor increased opportunistic infections, for example, by P. aeruginosa.
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Disbiosis/etiología , Microbioma Gastrointestinal/inmunología , Intestinos/microbiología , Pulmón/microbiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/inmunología , Vancomicina/toxicidad , Animales , Antibacterianos/toxicidad , Modelos Animales de Enfermedad , Disbiosis/patología , Heces/microbiología , Microbioma Gastrointestinal/efectos de los fármacos , Intestinos/efectos de los fármacos , Intestinos/inmunología , Pulmón/efectos de los fármacos , Pulmón/inmunología , Ratones , Ratones Endogámicos C57BL , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/efectos de los fármacosRESUMEN
Fever is a hallmark of infections and inflammatory diseases, represented by an increase of 1-4°C in core body temperature. Fever-range hyperthermia (FRH) has been shown to increase neutrophil recruitment to local sites of infection. Here, we evaluated the impact of a short period (1 h) of FRH (STFRH) on pro-inflammatory and bactericidal human neutrophil functions. STFRH did not affect neutrophil spontaneous apoptosis but reverted the lipopolysaccharide (LPS)-induced anti-apoptotic effect compared with that under normothermic conditions. Furthermore, STFRH accelerated phorbol myristate acetate (PMA)-induced NETosis evaluated either by the nuclear DNA decondensation at 2 h post-stimulation or by the increase in extracellular DNA that colocalized with myeloperoxidase (MPO) at 4 h post-stimulation. Increased NETosis upon STFRH was associated with an increase in reactive oxygen species (ROS) production but not in autophagy levels. STFRH also increased NETosis in response to Pseudomonas aeruginosa challenge but moderately reduced its phagocytosis. However, these STFRH-induced effects did not influence the ability of neutrophils to kill bacteria after 4 h of co-culture. STFRH also significantly reduced neutrophil capacity to release the pro-inflammatory cytokines chemokine (C-X-C motif) ligand 8/interleukin 8 (CXCL8/IL-8) and IL-1ß in response to LPS and P. aeruginosa challenge. Altogether, these results indicate that a short and mild hyperthermal period is enough to modulate neutrophil responses to bacterial encounter. They also suggest that fever spikes during bacterial infections might lead neutrophils to trigger an emergency response promoting neutrophil extracellular trap (NET) formation to ensnare bacteria in order to wall off the infection and to reduce their release of pro-inflammatory cytokines in order to limit the inflammatory response.
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Trampas Extracelulares/inmunología , Fiebre/inmunología , Interleucina-1beta/inmunología , Interleucina-8/inmunología , Neutrófilos/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Trampas Extracelulares/microbiología , Femenino , Fiebre/microbiología , Fiebre/patología , Humanos , Masculino , Neutrófilos/microbiología , Neutrófilos/patología , Infecciones por Pseudomonas/patologíaRESUMEN
Pseudomonas aeruginosa (Pa) detection in the paranasal sinuses may help to prevent or postpone bacterial aspiration to the lower airways (LAW) and chronic lung infection in cystic fibrosis (CF). We assessed the ability of an ELISA test for measurement of specific Pa secretory IgA (sIgA) in saliva (a potential marker of sinus colonization) to early detect changes in the Pa LAW status (indicated by microbiological sputum or cough swab culture and specific serum IgG levels) of 65 patients for three years, in different investigation scenarios. Increased sIgA levels were detected in saliva up to 22 months before changes in culture/serology. Patients who remained Pa-positive had significantly increased sIgA levels than patients who remained Pa-negative, both at the baseline (39.6 U/mL vs. 19.2 U/mL; p = 0.02) and at the end of the follow-up (119.4 U/mL vs. 25.2 U/mL; p < 0.001). No association was found between sIgA levels in saliva and emergence or recurrence of Pa in the LAW. A positive median sIgA result in the first year of follow-up implied up to 12.5-fold increased risk of subsequent Pa exposure in the LAW. Our test detected early changes in the P. aeruginosa LAW status and risk of exposure to P. aeruginosa in the LAW with two years in advance. Comparison with sinus culture is needed to assess the test's ability to identify CF patients in need of a sinus approach for Pa investigation, which could provide opportunities of Pa eradication before its aspiration to the lungs.
Asunto(s)
Fibrosis Quística/complicaciones , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoglobulina A Secretora/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Infecciones del Sistema Respiratorio/inmunología , Saliva/inmunología , Adolescente , Anticuerpos Antibacterianos/inmunología , Niño , Preescolar , Femenino , Estudios de Seguimiento , Humanos , Lactante , Masculino , Factores de TiempoRESUMEN
BACKGROUND Healthcare-associated infections caused by bacteria such as Pseudomonas aeruginosa are a major public health problem worldwide. Gene regulatory networks (GRN) computationally represent interactions among regulatory genes and their targets. They are an important approach to help understand bacterial behaviour and to provide novel ways of overcoming scientific challenges, including the identification of potential therapeutic targets and the development of new drugs. OBJECTIVES The goal of this study was to reconstruct the multidrug-resistant (MDR) P. aeruginosa GRN and to analyse its topological properties. METHODS The methodology used in this study was based on gene orthology inference using the reciprocal best hit method. We used the genome of P. aeruginosa CCBH4851 as the basis of the reconstruction process. This MDR strain is representative of the sequence type 277, which was involved in an endemic outbreak in Brazil. FINDINGS We obtained a network with a larger number of regulatory genes, target genes and interactions as compared to the previously reported network. Topological analysis results are in accordance with the complex network representation of biological processes. MAIN CONCLUSIONS The properties of the network were consistent with the biological features of P. aeruginosa. To the best of our knowledge, the P. aeruginosa GRN presented here is the most complete version available to date.
Asunto(s)
Humanos , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Infecciones por Pseudomonas/inmunología , Genes Reguladores/inmunología , Brasil/epidemiología , Genes MDR/genéticaRESUMEN
Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infections in immunocompromised individuals and in patients with cystic fibrosis. A range of vaccines to prevent infections caused by P. aeruginosa has already been tested, yet no vaccine against this pathogen is currently available. The goal of this study was to evaluate the potential of bovine serum albumin nanoparticles (BSA-NPs) associated with total P. aeruginosa ATCC 27853 antigens in inducing protection against the infection with virulent P. aeruginosa PA14 strain in murine model of nasal infection. Swiss mice were immunized with BSA-NPs associated with total P. aeruginosa antigens (NPPa) or empty NPs (NPe). As positive and negative control, groups of animals were immunized with total antigens of P. aeruginosa ATCC 27853 and phosphate buffered saline, respectively. Immunized mice were infected via nasal route using P. aeruginosa PA14 strain. The survival after 48â¯h was evaluated and the lungs from animals were processed for quantification of bacterial load, cytokine expression and histopathological analysis. After infection with P. aeruginosa PA14, animals immunized with NPPa had the highest survival rate, the lowest bacterial lung load, a controlled production of cytokines and few histopathological changes. These results indicate that NPPa immunization protected mice from infection, contributing for the elimination of the bacteria from the lungs, which consequently reflected the survival of the animals. Therefore, this vaccine was able to induce a functional response in an animal model of lethal infection and thereby is a promising platform for P. aeruginosa vaccines.
Asunto(s)
Nanopartículas/química , Infecciones por Pseudomonas/prevención & control , Vacunas contra la Infección por Pseudomonas/inmunología , Albúmina Sérica Bovina/química , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Carga Bacteriana , Citocinas/inmunología , Modelos Animales de Enfermedad , Femenino , Pulmón/microbiología , Ratones , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa , VacunaciónRESUMEN
BACKGROUND: Pseudomonas aeruginosa (PS) infection results in severe morbidity and mortality, especially in immune-deficient populations. Aerobic exercise (AE) modulates the immune system, but its effects on the outcomes of pulmonary PS infection in elderly mice are unknown. METHODS: BALB/c mice (24 weeks old) were randomized to sedentary, exercise (EX), PS, and PS + EX groups for the acute experimental setting, and PS and PS + EX groups for the chronic setting. Low-intensity AE was performed for 5 weeks, 60 min/day; 24 h after the final AE session, mice were inoculated with 5 × 104 colony-forming units (CFU) of PS, and 24 h and 14 days after PS inoculation, mice were studied. RESULTS: AE inhibited PS colonization (p < 0.001) and lung inflammation (total cells, neutrophils, lymphocytes [p < 0.01] in bronchoalveolar lavage [BAL]), with significant differences in BAL levels of IL-1ß (p < 0.001), IL-6 (p < 0.01), CXCL1 (p < 0.001), and TNF-α (p < 0.001), as well as parenchymal neutrophils (p < 0.001). AE increased BAL levels of IL-10 and parenchymal (p < 0.001) and epithelial (p < 0.001) IL-10 expression, while epithelial (p < 0.001) and parenchymal (p < 0.001) NF-κB expression was decreased. AE diminished pulmonary lipid peroxidation (p < 0.001) and increased glutathione peroxidase (p < 0.01). Pre-incubation of BEAS-2B with IL-10 inhibited PS-induced epithelial cell expression of TNF-α (p < 0.05), CD40 (p < 0.01), and dichlorodihydrofluorescein diacetate (p < 0.05). CONCLUSIONS: AE inhibits PS-induced lung inflammation and bacterial colonization in elderly mice, involving IL-10/NF-κB, and redox signaling.
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Ejercicio Físico/fisiología , Interleucina-10/metabolismo , Pulmón/inmunología , Neutrófilos/inmunología , Neumonía/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/fisiología , Envejecimiento , Animales , Modelos Animales de Enfermedad , Glutatión Peroxidasa/metabolismo , Humanos , Peroxidación de Lípido , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Condicionamiento Físico Animal , Neumonía/terapia , Infecciones por Pseudomonas/terapia , Transducción de SeñalRESUMEN
BACKGROUND AND OBJECTIVES: The mechanisms leading to low effectiveness of the humoral immune response against P. aeruginosa in cystic fibrosis (CF) are poorly understood. The aim of the present study was to assess the avidity maturation of specific antipseudomonal IgG before and during the development of chronic lung infection in a cohort of Danish CF patients. METHODS: Avidity maturation was assessed against a pooled P. aeruginosa antigen (St-Ag) and against P. aeruginosa alginate in 10 CF patients who developed chronic lung infection and 10 patients who developed intermittent lung colonization, using an ELISA technique with the thiocyanate elution method. Avidity was quantitatively determined by calculating the avidity Constant (Kav). RESULTS: IgG avidity to St-Ag significantly increased at the onset (Median Kav=2.47) and one year after the onset of chronic infection (Median Kav=3.27), but did not significantly changed in patients who developed intermittent colonization. IgG avidity against alginate did not significantly change over the years neither in patients who developed chronic lung infection (Median Kav=3.84 at the onset of chronic infection), nor in patients who developed intermittent colonization. CONCLUSION: IgG avidity to P. aeruginosa alginate does not significantly enhance as chronic lung infection progresses. This probably plays a role in the difficulty to mount an effective opsonophagocytic killing to clear mucoid P. aeruginosa infection in CF.
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Biopelículas , Fibrosis Quística , Inmunoglobulina G/sangre , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Adolescente , Adulto , Niño , Enfermedad Crónica , Fibrosis Quística/inmunología , Fibrosis Quística/microbiología , Dinamarca , Femenino , Humanos , Inmunidad Humoral , Masculino , Infecciones por Pseudomonas/diagnóstico , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/fisiopatología , Pseudomonas aeruginosa/aislamiento & purificación , Pseudomonas aeruginosa/fisiología , Reproducibilidad de los ResultadosRESUMEN
We assessed the diagnostic ability of an enzyme-linked immunosorbent assay test for measurement of specific secretory IgA (sIgA) in saliva to identify cystic fibrosis (CF) patients with Pseudomonas aeruginosa chronic lung infection and intermittent lung colonization. A total of 102 Brazilian CF patients and 53 healthy controls were included. Specific serum IgG response was used as a surrogate to distinguish CF patients according to their P. aeruginosa colonization/infection status. The rate of sIgA positivity was 87.1% in CF chronically infected patients (median value = 181.5 U/mL), 48.7% in intermittently colonized patients (median value = 45.8 U/mL) and 21.8% in free of infection patients (median value = 22.1 U/mL). sIgA levels in saliva were significantly associated with serum P. aeruginosa IgG and microbiological culture results. The sensitivity, specificity, PPV and NPV for differentiation between presence and absence of chronic lung infection were 87%, 63%, 51% and 92%, respectively. Measurement of sIgA in saliva may be used for screening patients in risk of developing P. aeruginosa chronic lung infection in CF and possibly also for paranasal sinusitis, and, most importantly, to efficiently rule out chronic P. aeruginosa lung infection.
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Anticuerpos Antibacterianos/análisis , Fibrosis Quística/diagnóstico , Inmunoglobulina A Secretora/análisis , Inmunoglobulina G/análisis , Infecciones Oportunistas/diagnóstico , Infecciones por Pseudomonas/diagnóstico , Saliva/química , Adolescente , Adulto , Estudios de Casos y Controles , Niño , Preescolar , Estudios Transversales , Fibrosis Quística/complicaciones , Fibrosis Quística/inmunología , Fibrosis Quística/microbiología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Pulmón/inmunología , Pulmón/microbiología , Pulmón/patología , Masculino , Infecciones Oportunistas/complicaciones , Infecciones Oportunistas/inmunología , Infecciones Oportunistas/microbiología , Infecciones por Pseudomonas/complicaciones , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa , Saliva/inmunología , Saliva/microbiología , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Sinonasal bitter taste receptors (T2Rs) contribute to upper airway innate immunity and correlate with chronic rhinosinusitis (CRS) clinical outcomes. A subset of T2Rs expressed on sinonasal solitary chemosensory cells (SCCs) are activated by denatonium, resulting in a calcium-mediated secretion of bactericidal antimicrobial peptides (AMPs) in neighboring ciliated epithelial cells. We hypothesized that there is patient variability in the amount of bacterial killing induced by different concentrations of denatonium and that the differences correlate with CRS clinical outcomes. METHODS: Bacterial growth inhibition was quantified after mixing bacteria with airway surface liquid (ASL) collected from denatonium-stimulated sinonasal air-liquid interface (ALI) cultures. Patient ASL bacterial killing at 0.1 mM denatonium and baseline characteristics and sinus surgery outcomes were compared between these populations. RESULTS: There is variability in the degree of denatonium-induced bacterial killing between patients. In CRS with nasal polyps (CRSwNP), patients with increased bacterial killing after stimulation with low levels of denatonium undergo significantly more functional endoscopic sinus surgeries (FESSs) (p = 0.037) and have worse 6-month post-FESS 22-item Sino-Nasal Outcome Test (SNOT-22) scores (p = 0.012). CONCLUSION: Bacterial killing after stimulation with low levels of denatonium correlates with number of prior FESS and postoperative SNOT-22 scores in CRSwNP. Some symptoms of CRS in patients with hyperresponsiveness to low levels of denatonium may be due to increased airway immune activity or inherent disease severity.
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Cilios/metabolismo , Pólipos Nasales/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/fisiología , Compuestos de Amonio Cuaternario/metabolismo , Rinitis/inmunología , Sinusitis/inmunología , Péptidos Catiónicos Antimicrobianos/metabolismo , Bacteriólisis , Señalización del Calcio , Procesos de Crecimiento Celular , Células Cultivadas , Enfermedad Crónica , Cilios/patología , Progresión de la Enfermedad , Endoscopía , Femenino , Humanos , Inmunidad Innata , Masculino , Pólipos Nasales/microbiología , Rinitis/microbiología , Sinusitis/microbiología , Resultado del TratamientoRESUMEN
Two-component systems are widespread in bacteria, allowing adaptation to environmental changes. The classical pathway is composed of a histidine kinase that phosphorylates an aspartate residue in the cognate response regulator (RR). RRs lacking the phosphorylatable aspartate also occur, but their function and contribution during host-pathogen interactions are poorly characterized. AtvR (PA14_26570) is the only atypical response regulator with a DNA-binding domain in the opportunistic pathogen Pseudomonas aeruginosa Macrophage infection with the atvR mutant strain resulted in higher levels of tumor necrosis factor alpha secretion as well as increased bacterial clearance compared to those for macrophages infected with the wild-type strain. In an acute pneumonia model, mice infected with the atvR mutant presented increased amounts of proinflammatory cytokines, increased neutrophil recruitment to the lungs, reductions in bacterial burdens, and higher survival rates in comparison with the findings for mice infected with the wild-type strain. Further, several genes involved in hypoxia/anoxia adaptation were upregulated upon atvR overexpression, as seen by high-throughput transcriptome sequencing (RNA-Seq) analysis. In addition, atvR was more expressed in hypoxia in the presence of nitrate and required for full expression of nitrate reductase genes, promoting bacterial growth under this condition. Thus, AtvR would be crucial for successful infection, aiding P. aeruginosa survival under conditions of low oxygen tension in the host. Taken together, our data demonstrate that the atypical response regulator AtvR is part of the repertoire of transcriptional regulators involved in the lifestyle switch from aerobic to anaerobic conditions. This finding increases the complexity of regulation of one of the central metabolic pathways that contributes to Pseudomonas ubiquity and versatility.
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Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Anaerobiosis , Animales , Carga Bacteriana , Citocinas/biosíntesis , Citocinas/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento , Interacciones Huésped-Patógeno , Hipoxia , Pulmón/inmunología , Macrófagos/microbiología , Ratones , Mutación , Neutrófilos/inmunología , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/microbiología , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/fisiología , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , VirulenciaRESUMEN
Selenoprotein T (SELENOT) is an endoplasmatic reticulum (ER)-associated redoxin that contains the amino acid selenocysteine (Sec, U) within a CXXU motif within a thioredoxin-like fold. Its precise function in multicellular organisms is not completely understood although it has been shown in mammals to be involved in Ca2+ homeostasis, antioxidant and neuroendocrine functions. Here, we use the model organism C. elegans to address SELENOT function in a whole organism throughout its life cycle. C. elegans possess two genes encoding SELENOT protein orthologues (SELT-1.1 and SELT-1.2), which lack Sec and contain the CXXC redox motif instead. Our results show that a SecâCys replacement and a gene duplication were two major evolutionary events that occurred in the nematode lineage. We find that worm SELT-1.1 localizes to the ER and is expressed in different cell types, including the nervous system. In contrast, SELT-1.2 exclusively localizes in the cytoplasm of the AWB neurons. We find that selt-1.1 and selt-1.2 single mutants as well as the double mutant are viable, but the selt-1.1 mutant is compromised under rotenone-induced oxidative stress. We demonstrate that selt-1.1, but not selt-1.2, is required for avoidance to the bacterial pathogens Serratia marcescens and Pseudomonas aeruginosa. Aversion to the noxious signal 2-nonanone is also significantly impaired in selt-1.1, but not in selt-1.2 mutant animals. Our results suggest that selt-1.1 would be a redox transducer required for nociception and optimal organismal fitness. The results highlight C. elegans as a valuable model organism to study SELENOT-dependent processes.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/inmunología , Retículo Endoplásmico/metabolismo , Neuronas/metabolismo , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Selenoproteínas/metabolismo , Infecciones por Serratia/inmunología , Serratia marcescens/inmunología , Animales , Proteínas de Caenorhabditis elegans/genética , Células Cultivadas , Cisteína/genética , Duplicación de Gen , Inmunidad Innata , Cetonas/administración & dosificación , Estadios del Ciclo de Vida , Mutación/genética , Nocicepción , Estrés Oxidativo , Transporte de Proteínas , Selenoproteínas/genéticaRESUMEN
For opportunistic pathogens such as Pseudomonas aeruginosa, the mucosal barrier represents a formidable challenge. Infections develop only in patients with altered epithelial barriers. Here, we showed that P. aeruginosa interacts with a polarized epithelium, adhering almost exclusively at sites of multi-cellular junctions. In these sites, numerous bacteria attach to an extruded apoptotic cell or apoptotic body. This dead cell tropism is independent of the type of cell death, as P. aeruginosa also binds to necrotic cells. We further showed that P. aeruginosa is internalized through efferocytosis, a process in which surrounding epithelial cells engulf and dispose of extruded apoptotic cells. Intracellularly, along with apoptotic cell debris, P. aeruginosa inhabits an efferocytic phagosome that acquires lysosomal features, and is finally killed. We propose that elimination of P. aeruginosa through efferocytosis is part of a host defense mechanism. Our findings could be relevant for the study of cystic fibrosis, which is characterized by an exacerbated number of apoptotic cells and ineffective efferocytosis.
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Apoptosis , Células Epiteliales/microbiología , Fagocitosis/inmunología , Infecciones por Pseudomonas/inmunología , Animales , Línea Celular , Perros , Humanos , Procesamiento de Imagen Asistido por Computador , Células de Riñón Canino Madin Darby , Microscopía Electrónica de Transmisión , Pseudomonas aeruginosa/inmunologíaRESUMEN
AIMS: Recurrent infections and activation of the inflammatory response affect the prognosis of cystic fibrosis (CF). We investigated the relationship between inflammatory response, infection, and pulmonary function in CF. MAIN METHODS: A clinical-cross-sectional study was conducted with 86 subjects: control group (CG, n=31, the same age and sex of the CF group), and CF group (CFG, n=55, age: 1-16 years), further distributed into CFG negative or positive bacteriology (CFGB(-)/CFGB(+)), and CFG negative or positive Pseudomonas aeruginosa (CFGPa(-)/CFGPa(+)). Using the Wald test, multiple linear regression (95% confidence interval) was performed between CG and CFG, and between CG and each of the CF subgroups (CFGB(-)/CFGB(+) and CFGPa(-)/CFGPa(+)). The inflammatory markers evaluated were myeloperoxidase (MPO), adenosine deaminase (ADA) activities, interleukin-1beta (IL-1ß), tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP), nitric oxide metabolites (NOx) levels, and total and differential leukocyte counts. KEY FINDINGS: After adjusting for sex and age, CFG compared to CG revealed an increase of MPO, IL-1ß (P<0.001 in all subgroups), and CRP: CFG (P=0.002), CFGB(-) (P=0.007), CFGB(+) (P=0.009), CFGPa(-) (P=0.004) and CFGPa(+) (P=0.020). NOx (P=0.001, P<0.001), leukocytes (P=0.002, P=0.001), and neutrophils (P=0.003, P<0.001) were increased in CFGB(+) and CFGPa(+), respectively. A negative correlation between FEV1 and leukocytes (P=0.008) and FEV1 and neutrophils (P=0.031) resulted in CFG. SIGNIFICANCE: The inflammatory response characterized by the increase of MPO, IL-1ß, and CRP is determinant for CF. Also leukocytosis due to neutrophilia determines the pulmonary function deficiency in this disease.
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Fibrosis Quística/complicaciones , Neumonía/complicaciones , Infecciones por Pseudomonas/complicaciones , Pseudomonas aeruginosa/inmunología , Adolescente , Niño , Preescolar , Estudios Transversales , Fibrosis Quística/diagnóstico , Fibrosis Quística/inmunología , Fibrosis Quística/microbiología , Femenino , Humanos , Lactante , Pulmón/inmunología , Pulmón/microbiología , Masculino , Neumonía/diagnóstico , Neumonía/inmunología , Neumonía/microbiología , Infecciones por Pseudomonas/diagnóstico , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/aislamiento & purificaciónRESUMEN
Pseudomonas aeruginosa is an important opportunistic human pathogen that causes severe infections in immunocompromised patients and also in cystic fibrosis patients. The aim of this work was to study if a bovine serum albumin nanoparticles with entrapped antigens extracted from P. aeruginosa would be able to protect mice from nasal infection by this pathogen. Mice were immunized via the subcutaneous route using P. aeruginosa antigens, empty nanoparticles or nanoparticles with entrapped P. aeruginosa antigens on days 0, 7 and 14. The total IgG antibody production and specific IgG1 and IgG2a titer were measured by ELISA. Immunized mice were challenged with live P. aeruginosa and their lungs were collected for histopathology studies. Our data showed that NPPa-vaccinated mice presented a high anti-Pseudomonas IgG1 and a low IgG2a antibody titles and decreased inflammatory signs, with significant reduction in intensity and concentration of inflammatory cells, lower hemorrhagic, edema and hyperemia signs in the lungs of challenge mice with live P. aeruginosa if compared to the other groups. Therefore, this formulation is able to induce a functional response in an animal model of infection and thereby is a promising platform for P. aeruginosa vaccines.
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Antígenos Bacterianos/inmunología , Vacunas Bacterianas/inmunología , Pulmón/patología , Nanopartículas , Infecciones por Pseudomonas/prevención & control , Albúmina Sérica Bovina/administración & dosificación , Animales , Anticuerpos Antibacterianos/sangre , Inmunoglobulina G/sangre , Inflamación/microbiología , Inflamación/patología , Pulmón/microbiología , Masculino , Ratones , Infecciones por Pseudomonas/inmunologíaRESUMEN
Pseudomonas aeruginosa is associated with increased mortality in cystic fibrosis (CF) patients, and expresses type III secretion system proteins (TTSP), which is a common mechanism used by gram-negative pathogens for delivery of anti-host factors. Our aim was to investigate whether or not these antigens (TTSP) would be recognized by CF sera, by Western blot reaction. We have showed herein that all patients (n = 11) not chronically infected by P. aeruginosa had their first serum positive for TTSP (ExoS, ExoT, PopB, and/or PopD). All chronic patients had a strong positive serology to TTSP, although relatively weak reactions to TTSP were observed for some individuals in the negative control group. Therefore, TTSP that were early produced in P. aeruginosa infected CF patients, induced a detectable antibody response in those patients and were easily detected by Western-blot reaction.
Asunto(s)
Fibrosis Quística/microbiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/aislamiento & purificación , Infecciones del Sistema Respiratorio/microbiología , Adolescente , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/sangre , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/sangre , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Niño , Preescolar , Estudios Transversales , Fibrosis Quística/sangre , Fibrosis Quística/metabolismo , Humanos , Infecciones por Pseudomonas/sangre , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/metabolismo , Estudios RetrospectivosRESUMEN
Pathogenic bacteria delay wound healing through several different mechanisms such as persistent production of inflammatory mediators or maintenance of necrotic neutrophils, which release cytolytic enzymes and free oxygen radicals. One of the most frequent pathogens isolated from infections in chronic wounds is Pseudomonas aeruginosa. This bacterium is extremely refractory to therapy and to host immune attack when it forms biofilms. Therefore, antibiotics and antiseptics are becoming useless in the treatment of these infections. In previous works, we demonstrated that Lactobacillus plantarum has an important antipathogenic capacity on P. aeruginosa. The aim of the present work was to elucidate the mechanism involved in the control of growth of P. aeruginosa on different surfaces by L. plantarum. For this purpose, we investigated the effects of L. plantarum supernatants on pathogenic properties of P. aeruginosa, such as adhesion, viability, virulence factors, biofilm formation, and quorum sensing signal expression. L. plantarum supernatants were able to inhibit pathogenic properties of P. aeruginosa by a quorum quenching mechanism. The antipathogenic properties mentioned above, together with the immunomodulatory, tissue repair, and angiogenesis properties in the supernatants of L. plantarum, make them an attractive option in infected chronic wound treatment.