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1.
Microbiology (Reading) ; 170(8)2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-39177453

RESUMEN

Escherichia coli (E. coli) is a major cause of urinary tract infections, bacteraemia, and sepsis. CFT073 is a prototypic, urosepsis isolate of sequence type (ST) 73. This laboratory, among others, has shown that strain CFT073 is resistant to serum, with capsule and other extracellular polysaccharides imparting resistance. The interplay of such polysaccharides remains under-explored. This study has shown that CFT073 mutants deficient in lipopolysaccharide (LPS) O-antigen and capsule display exquisite serum sensitivity. Additionally, O-antigen and LPS outer core mutants displayed significantly decreased surface K2 capsule, coupled with increased unbound K2 capsule being detected in the supernatant. The R1 core and O6 antigen are involved in the tethering of K2 capsule to the CFT073 cell surface, highlighting the importance of the R1 core in serum resistance. The dependence of capsule on LPS was shown to be post-transcriptional and related to changes in cell surface hydrophobicity. Furthermore, immunofluorescence microscopy suggested that the surface pattern of capsule is altered in such LPS core mutants, which display a punctate capsule pattern. Finally, targeting LPS biosynthesis using sub-inhibitory concentrations of a WaaG inhibitor resulted in increased serum sensitivity and decreased capsule in CFT073. Interestingly, the dependency of capsule on LPS has been observed previously in other Enterobacteria, indicating that the synergy between these polysaccharides is not just strain, serotype or species-specific but may be conserved across several pathogenic Gram-negative species. Therefore, using WaaG inhibitor derivatives to target LPS is a promising therapeutic strategy to reduce morbidity and mortality by reducing or eliminating surface capsule.


Asunto(s)
Cápsulas Bacterianas , Lipopolisacáridos , Lipopolisacáridos/metabolismo , Cápsulas Bacterianas/metabolismo , Cápsulas Bacterianas/genética , Humanos , Escherichia coli Patógena Extraintestinal/genética , Escherichia coli Patógena Extraintestinal/efectos de los fármacos , Escherichia coli Patógena Extraintestinal/metabolismo , Antígenos O/genética , Antígenos O/metabolismo , Infecciones por Escherichia coli/microbiología , Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutación
2.
Health Promot Pract ; 20(5): 703-710, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-30701986

RESUMEN

Objectives. Distracted driving is a major public health issue in the United States. In response to requests from high school students participating in a university-based initiative, the authors describe the collaborative development and implementation of a curriculum designed to address distracted driving behaviors among students in four high-needs school districts in the northeastern United States. Method. The curriculum integrates current statistics on distracted and drowsy driving and three interactive learning stations: driving while distracted, walking while distracted, and driving while drowsy. Pre- and postsurveys were conducted to collect student driving data, assess student satisfaction with the program, and assess their likelihood of speaking up as a passenger in a high-risk situation. Results. The majority of students reported that they learned new information and would recommend the program to others. A Wilcoxon signed-rank test showed that students were more likely to speak up as a passenger with a distracted or drowsy driver (p < .001) after the program. Conclusion. This experience demonstrates a voluntary, multidisciplinary, university-based collaboration in the development of a novel public health education initiative. Based on the success of this phase, school districts elected to participate in Train the Trainer sessions to continue the program within their local high-needs school district.


Asunto(s)
Conducción Distraída/psicología , Educación en Salud/organización & administración , Estudiantes/psicología , Adolescente , Comportamiento del Consumidor , Curriculum , Femenino , Humanos , Masculino , Somnolencia , Estados Unidos , Caminata
3.
Crit Care Med ; 46(8): e805-e810, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29782355

RESUMEN

OBJECTIVES: The vascular endothelium is a major target of sepsis-induced events, and endothelial activation accounts for much of the pathology of sepsis. Urinary tract infections and pneumonia caused by Escherichia coli are among of the most common infections causing sepsis in both community and hospital settings. Currently, there are no approved drugs on the market to treat the underlying pathophysiology of sepsis. The aim of this study is to elucidate the molecular mechanism by which E. coli induces endothelial injury as a result of attachment. DESIGN: Laboratory research using a hemodynamic perfusion ex vivo model. SETTING: Research Laboratories of Royal College of Surgeons in Ireland and Beaumont Hospital. PATIENTS: Ex vivo human vascular endothelial cells. INTERVENTIONS: Addition of αVß3 antagonist, cilengitide. MEASUREMENTS AND MAIN RESULTS: Clinical strains of E. coli isolated from patients with sepsis bound to sheared human endothelial cells under static and hemodynamic shear conditions. Binding was dependent on E. coli cell membrane protein outer membrane protein A attaching directly to endothelial cell integrin αVß3. Attachment resulted in disturbances in endothelial barrier integrity, as determined by loss of tight junction protein staining, permeability changes, and ultimately cell death by apoptosis. Using a low concentration of the αVß3 antagonist cilengitide or using a strain deficient in outer membrane protein A resulted in a significant reduction in endothelial dysfunction following infection. CONCLUSIONS: Inhibition of E. coli binding to endothelial cell αVß3 by cilengitide prevents endothelial dysfunction and may, therefore, present as a novel early therapeutic for the treatment of sepsis.


Asunto(s)
Células Endoteliales/microbiología , Escherichia coli/crecimiento & desarrollo , Integrina alfaVbeta3/antagonistas & inhibidores , Sepsis/microbiología , Venenos de Serpiente/farmacología , Permeabilidad Capilar , Relación Dosis-Respuesta a Droga , Humanos
4.
Pain Med ; 19(7): 1425-1435, 2018 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-29474648

RESUMEN

OBJECTIVE: Despite the high prevalence of chronic multisite pain, there is little consensus on methods to characterize it. Commonly used assessments report only one dimension of pain, that is, intensity, thus ignoring the spatial aspect of pain. We developed a novel pain quantification index, the Integrated Pain Quantification Index (IPQI), on a scale of 0 to 1 that integrates multiple distinct pain measures into a single value, thus representing multidimensional pain information with a single value. DESIGN: Single-visit, noninterventional, epidemiological study. SETTING: Fourteen outpatient multidisciplinary pain management programs. PATIENTS: Patients with chronic pain of the trunk and/or limbs for at least six months with average overall pain intensity of at least 5 on the numeric rating scale. METHODS: Development of IPQI was performed in a large population (N = 810) of chronic pain patients from the Multiple Areas of Pain (MAP) study. RESULTS: Prevalence of two or more noncontiguous painful areas was at 88.3% (95% confidence interval [CI] = 0.86-0.90), with a mean of 6.3 areas (SD = 5.57 areas). Prevalence of more than 10% body area in pain was at 52.8% (95% CI = 0.49-0.56), with a mean at 16.1% (17.16%). On average, IPQI values were near the middle of the scale, with mean and median IPQI at 0.52 (SD = 0.13) and 0.55, respectively. The IPQI was generalizable and clinically relevant across all domains recommended by the Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials. CONCLUSIONS: IPQI provided a single pain score for representing complex, multidimensional pain information on one scale and has implications for comparing pain populations across longitudinal clinical trials.


Asunto(s)
Dolor Crónico/diagnóstico , Dimensión del Dolor/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Encuestas y Cuestionarios , Adulto Joven
5.
N Engl J Med ; 366(21): 1978-86, 2012 May 24.
Artículo en Inglés | MEDLINE | ID: mdl-22607135

RESUMEN

BACKGROUND: Women with cystic fibrosis are at increased risk for mucoid conversion of Pseudomonas aeruginosa, which contributes to a sexual dichotomy in disease severity. METHODS: We evaluated the effects of estradiol and its metabolite estriol on P. aeruginosa in vitro and in vivo and determined the effect of estradiol on disease exacerbations in women with cystic fibrosis. RESULTS: Estradiol and estriol induced alginate production in P. aeruginosa strain 01 and in clinical isolates obtained from patients with and those without cystic fibrosis. After prolonged exposure to estradiol, P. aeruginosa adopted early mucoid morphology, whereas short-term exposure inhibited bacterial catalase activity and increased levels of hydrogen peroxide, which is potentially damaging to DNA. Consequently, a frameshift mutation was identified in mucA, a key regulator of alginate biosynthesis in P. aeruginosa. In vivo levels of estradiol correlated with infective exacerbations in women with cystic fibrosis, with the majority occurring during the follicular phase (P<0.05). A review of the Cystic Fibrosis Registry of Ireland revealed that the use of oral contraceptives was associated with a decreased need for antibiotics. Predominantly nonmucoid P. aeruginosa was isolated from sputum during exacerbations in the luteal phase (low estradiol). Increased proportions of mucoid bacteria were isolated during exacerbations occurring in the follicular phase (high estradiol), with a variable P. aeruginosa phenotype evident in vivo during the course of the menstrual cycle corresponding to fluctuating estradiol levels. CONCLUSIONS: Estradiol and estriol induced mucoid conversion of P. aeruginosa in women with cystic fibrosis through a mutation of mucA in vitro and were associated with selectivity for mucoid isolation, increased exacerbations, and mucoid conversion in vivo. (Funded by the Molecular Medicine Ireland Clinician-Scientist Fellowship Programme.).


Asunto(s)
Estradiol/farmacología , Estriol/farmacología , Polisacáridos Bacterianos/biosíntesis , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/efectos de los fármacos , Alginatos , Fibrosis Quística/microbiología , Estradiol/uso terapéutico , Femenino , Regulación Bacteriana de la Expresión Génica , Ácido Glucurónico/biosíntesis , Ácido Glucurónico/genética , Ácidos Hexurónicos , Humanos , Irlanda , Fenotipo , Polisacáridos Bacterianos/genética , Embarazo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Sistema de Registros
6.
Nucleic Acids Res ; 41(6): e71, 2013 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-23325846

RESUMEN

MicroRNAs (miRNAs) are small non-coding RNAs that regulate expression by translational repression or messenger RNA (mRNA) degradation. Although numerous bioinformatic prediction models exist to identify miRNA-mRNA interactions, experimental validation of bona fide interactions can be difficult and laborious. Few methods can comprehensively identify miRNAs that target a single mRNA. We have developed an experimental approach to search for miRNAs targeting any mRNA using a capture affinity assay involving a biotinylated DNA anti-sense oligonucleotide. This method identifies miRNAs targeting the full length of the mRNA. The method was tested using three separate mRNA targets: alpha-1 antitrypsin (AAT) mRNA, interleukin-8 mRNA and secretory leucoprotease inhibitor mRNA. AAT mRNA-specific and total miRNAs from three different cell lines (monocytic THP-1, bronchial epithelial 16HBE14o- and liver HepG2 cells) were profiled, and validation studies revealed that AAT mRNA-specific miRNAs functionally target the AAT mRNA in a cell-specific manner, providing the first evidence of innate miRNAs selectively targeting and modulating AAT mRNA expression. Interleukin-8 and secretory leucoprotease inhibitor mRNAs and their cognate miRNAs were also successfully captured using this approach. This is a simple and an efficient method to potentially identify miRNAs targeting sequences within the full length of a given mRNA transcript.


Asunto(s)
MicroARNs/aislamiento & purificación , Oligodesoxirribonucleótidos Antisentido , ARN Mensajero/aislamiento & purificación , Sitios de Unión , Biotinilación , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Humanos , Interleucina-8/genética , MicroARNs/metabolismo , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inhibidor Secretorio de Peptidasas Leucocitarias/genética , Transcriptoma , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo
7.
Infect Immun ; 82(1): 298-305, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24166954

RESUMEN

Extraintestinal Escherichia coli (ExPEC) organisms are the leading cause of Gram-negative bacterial bloodstream infections. These bacteria adapt to survival in the bloodstream through expression of factors involved in scavenging of nutrients and resisting the killing activity of serum. In this study, the transcriptional response of a prototypic ExPEC strain (CFT073) to human serum was investigated. Resistance of CFT073 to the bactericidal properties of serum involved increased expression of envelope stress regulators, including CpxR, σE, and RcsB. Many of the upregulated genes induced by active serum were regulated by the Rcs two-component system. This system is triggered by envelope stress such as changes to cell wall integrity. RcsB-mediated serum resistance was conferred through induction of the exopolysaccharide colanic acid. Production of this exopolysaccharide may be protective while cell wall damage caused by serum components is repaired.


Asunto(s)
Actividad Bactericida de la Sangre , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Polisacáridos/metabolismo , Actividad Bactericida de la Sangre/inmunología , Proteínas del Sistema Complemento/metabolismo , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Humanos , Análisis por Micromatrices , Estrés Fisiológico/fisiología
8.
Noncoding RNA ; 9(1)2023 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-36649035

RESUMEN

Long non-coding RNAs (lncRNAs) regulate gene expression. Their expression in alpha-1 antitrypsin (AAT) deficiency has not been investigated. Treatment of AAT deficiency involves infusion of plasma-purified AAT and this augmentation therapy has previously been shown to alter microRNA expression in monocytes of AAT-deficient (ZZ) individuals. Here, we assess the effect of AAT augmentation therapy on the lncRNA expression profile in ZZ monocytes. Peripheral blood monocytes were isolated from ZZ individuals pre (Day 0)- and post (Day 2)-AAT augmentation therapy. Arraystar lncRNA microarray profiling was performed; a total of 17,761 lncRNAs were detectable across all samples. The array identified 7509 lncRNAs with differential expression post-augmentation therapy, 3084 were increased and 4425 were decreased (fold change ≥ 2). Expression of many of these lncRNAs were similarly altered in ZZ monocytes treated ex vivo with 27.5 µM AAT for 4 h. These properties may contribute to the manifold effects of AAT augmentation therapy.

9.
Pain Manag ; 13(2): 115-127, 2023 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-36691862

RESUMEN

Aim: The availability of long-term (>2 years) safety outcomes of spinal cord stimulation (SCS) remains limited. We evaluated safety in a global SCS registry for chronic pain. Methods: Participants were prospectively enrolled globally at 79 implanting centers and followed out to 3 years after device implantation. Results: Of 1881 participants enrolled, 1289 received a permanent SCS implant (1776 completed trial). The annualized rate of device explant was 3.5% (all causes), and 1.1% due to inadequate pain relief. Total incidence of device explantation >3 years was 7.6% (n = 98). Of these, 32 subjects (2.5%) indicated inadequate pain relief as cause for removal. Implant site infection (11 events) was the most common device-related serious adverse event (<1%). Conclusion: This prospective, global, real-world study demonstrates a high-level of safety for SCS with low rate of explant/serious adverse events. Clinical Trial Registration: NCT01719055 (ClinicalTrials.gov).


Asunto(s)
Dolor Crónico , Estimulación de la Médula Espinal , Humanos , Estimulación de la Médula Espinal/efectos adversos , Estudios Prospectivos , Dolor Crónico/terapia , Complicaciones Posoperatorias , Sistema de Registros , Médula Espinal , Resultado del Tratamiento
10.
J Immunol ; 184(3): 1642-52, 2010 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-20026745

RESUMEN

Dysregulation of airway inflammation contributes to lung disease in cystic fibrosis (CF). Inflammation is mediated by inflammatory cytokines, including IL-8, which illustrates an increase in biological half-life and proinflammatory activity when bound to glycosaminoglycans (GAGs). The aim of this project was to compare IL-8 and IL-18 for their relative stability, activity, and interaction with GAGs, including chondroitin sulfate, hyaluronic acid, and heparan sulfate, present in high quantities in the lungs of patients with CF. Bronchoalveolar lavage fluid was collected from patients with CF (n = 28), non-CF controls (n = 14), and patients with chronic obstructive pulmonary disease (n = 12). Increased levels of IL-8 and reduced concentrations of IL-18 were detected in bronchial samples obtained from CF individuals. The low level of IL-18 was not a defect in IL-18 production, as the pro- and mature forms of the molecule were expressed and produced by CF epithelial cells and monocytes. There was, however, a marked competition between IL-8 and IL-18 for binding to GAGs. A pronounced loss of IL-18 binding capacity occurred in the presence of IL-8, which displaced IL-18 from these anionic-matrices, rendering the cytokine susceptible to proteolytic degradation by neutrophil elastase. As a biological consequence of IL-18 degradation, reduced levels of IL-2 were secreted by Jurkat T lymphocytes. In conclusion, a novel mechanism has been identified highlighting the potential of IL-8 to determine the fate of other inflammatory molecules, such as IL-18, within the inflammatory milieu of the CF lung.


Asunto(s)
Fibrosis Quística/inmunología , Fibrosis Quística/metabolismo , Glicosaminoglicanos/metabolismo , Interleucina-18/química , Interleucina-18/metabolismo , Interleucina-8/fisiología , Adolescente , Unión Competitiva/inmunología , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Línea Celular Transformada , Niño , Preescolar , Fibrosis Quística/patología , Regulación hacia Abajo/inmunología , Glicosaminoglicanos/química , Humanos , Mediadores de Inflamación/química , Mediadores de Inflamación/metabolismo , Interleucina-8/biosíntesis , Células Jurkat , Unión Proteica/inmunología , Estabilidad Proteica , Regulación hacia Arriba/inmunología
11.
Respir Care ; 67(10): 1254-1263, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-35728825

RESUMEN

BACKGROUND: Workforce development for the respiratory therapy (RT) profession is a growing concern. Upcoming staffing difficulties are expected due to retirement, attrition from the profession, and decreased enrollment in accredited RT programs nationwide. This study assessed respiratory therapists' perceptions of staffing needs and future trajectory of the RT profession. METHODS: This cross-sectional study utilized a modified 39-question survey tool delivered via e-mail to 618 Louisiana members of the American Association for Respiratory Care (AARC) between November 2019-February 2020. RESULTS: The response rate was 19% (118/618). Although 50% of respondents perceived understaffing, 77.6% indicated the importance to remain in the RT profession. A majority (93.1%) agreed on the importance of maintaining an active membership in the AARC. Respondents working in a hospital setting perceived understaffed work environments more often than other groups. Salary was most important to the employee (33.6%, 39/116), followed equally by room for growth (14.7%, 17/116) and scope of practice (14.7%, 17/116). For the future of the profession, the ability to assess patients and develop care plans and the ability to receive reimbursement for services were indicated as most important factors. Most (69.8%) agreed that the entry-level minimum should be increased to the bachelor's degree, and 21.6% agreed the master's degree in RT should be supported to increase scope of practice. CONCLUSIONS: This study indicated a consistent perception of understaffed work environments in respiratory care, and respondents expressed a perceived importance of remaining in the RT profession. This study also indicated support for raising the entry-level standard in RT and a desire for higher education to achieve professional growth and advancement.


Asunto(s)
Pandemias , Terapia Respiratoria , Estudios Transversales , Humanos , Terapia Respiratoria/educación , Encuestas y Cuestionarios , Estados Unidos , Recursos Humanos
12.
Microbiology (Reading) ; 157(Pt 4): 1042-1049, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21292751

RESUMEN

Biofilm production by staphylococci is an important virulence determinant mediated by the icaADBC-encoded polysaccharide intercellular adhesin (PIA) or by surface and extracellular proteins. Deletion of the Staphylococcus accessory regulator sarX significantly reduced biofilm-forming capacity in Staphylococcus epidermidis CSF41498, whereas multicopy sarX complemented the sarX mutant and increased wild-type biofilm production. In Staphylococcus aureus, SarX negatively regulates the accessory gene regulator (Agr) system, which in turn has strain-specific effects on biofilm regulation. Here we found that purified S. epidermidis SarX protein bound specifically to the agr P3 promoter. However RT-PCR analysis revealed that both mutation of sarX and multicopy sarX activated RNAIII transcription, making it difficult to correlate sarX-mediated biofilm regulation with altered agr activity. In contrast, RT-PCR and immunoblot analysis revealed that icaA transcription and PIA expression were decreased in the sarX mutant, whereas multicopy sarX increased ica and PIA expression. Furthermore, multicopy sarX did not promote biofilms in an icaC mutant. Finally, purified SarX protein bound specifically to the ica operon promoter. Taken together, these data reveal that the S. epidermidis SarX protein regulates the transcriptional activity of the agr and ica loci and controls the biofilm phenotype, primarily by regulating icaADBC transcription and PIA production.


Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Polisacáridos Bacterianos/biosíntesis , Staphylococcus epidermidis/fisiología , Factores de Transcripción/metabolismo , Proteínas Bacterianas/aislamiento & purificación , ADN Bacteriano/metabolismo , Eliminación de Gen , Perfilación de la Expresión Génica , Prueba de Complementación Genética , Immunoblotting , Regiones Promotoras Genéticas , Unión Proteica , ARN Bacteriano/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/aislamiento & purificación , Transcripción Genética
13.
J Bacteriol ; 192(21): 5822-31, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20802035

RESUMEN

In most cases, Escherichia coli exists as a harmless commensal organism, but it may on occasion cause intestinal and/or extraintestinal disease. Enterotoxigenic E. coli (ETEC) is the predominant cause of E. coli-mediated diarrhea in the developing world and is responsible for a significant portion of pediatric deaths. In this study, we determined the complete genomic sequence of E. coli H10407, a prototypical strain of enterotoxigenic E. coli, which reproducibly elicits diarrhea in human volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains, revealing that the chromosome is closely related to that of the nonpathogenic commensal strain E. coli HS and to those of the laboratory strains E. coli K-12 and C. Furthermore, these analyses demonstrated that there were no chromosomally encoded factors unique to any sequenced ETEC strains. Comparison of the E. coli H10407 plasmids with those from several ETEC strains revealed that the plasmids had a mosaic structure but that several loci were conserved among ETEC strains. This study provides a genetic context for the vast amount of experimental and epidemiological data that have been published.


Asunto(s)
Escherichia coli Enterotoxigénica/clasificación , Escherichia coli Enterotoxigénica/genética , Proteínas de Escherichia coli/metabolismo , Genoma Bacteriano , Secuencia de Aminoácidos , Cromosomas Bacterianos , Proteínas de Escherichia coli/genética , Proteínas Fimbrias , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica/fisiología , Datos de Secuencia Molecular , Plásmidos/genética
14.
Microbiology (Reading) ; 156(Pt 9): 2796-2806, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20507887

RESUMEN

The pathogenesis of diarrhoeal disease due to human enterotoxigenic Escherichia coli absolutely requires the expression of fimbriae. The expression of CS1 fimbriae is positively regulated by the AraC-like protein Rns. AraC-like proteins are DNA-binding proteins that typically contain two helix-turn-helix (HTH) motifs. A program of pentapeptide insertion mutagenesis of the Rns protein was performed, and this revealed that both HTH motifs are required by Rns to positively regulate CS1 fimbrial gene expression. Intriguingly, a pentapeptide insertion after amino acid C102 reduced the ability of Rns to transactivate CS1 fimbrial expression. The structure of Rns in this vicinity (NACRS) was predicted to be disordered and thus might act as a flexible linker. This hypothesis was confirmed by deletion of this amino acid sequence from the Rns protein; a truncated protein that lacked this sequence was no longer functional. Strikingly, this sequence could be functionally substituted in vivo and in vitro by a flexible seven amino acid sequence from another E. coli AraC-like protein RhaS. Our data indicate that HTH motifs and a flexible sequence are required by Rns for maximal activation of fimbrial gene expression.


Asunto(s)
Escherichia coli Enterotoxigénica/genética , Genes Reguladores , Mutagénesis , Transactivadores/química , Transactivadores/metabolismo , Secuencia de Aminoácidos , Escherichia coli Enterotoxigénica/química , Escherichia coli Enterotoxigénica/metabolismo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Secuencias Hélice-Giro-Hélice , Datos de Secuencia Molecular , Eliminación de Secuencia , Transactivadores/genética
15.
J Clin Microbiol ; 48(4): 1099-104, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20107091

RESUMEN

Escherichia coli is a major cause of bloodstream infections and death due to sepsis. It is the most frequent Gram-negative bacterial pathogen recovered from cultures of blood from both community-acquired and nosocomial cases. We set out to determine the relationships between E. coli virulence factors (VFs), phylogenetic groups, and antibiotic resistance and whether bacteremia cases had a community, health care-associated. or nosocomial origin. Isolates from consecutive episodes of E. coli bacteremia in 303 patients presenting to a university hospital were screened for their VFs, phylogenetic group, and antibiotic resistance. The majority of VFs present in the collection were equally distributed between antibiotic-susceptible and multiple-drug-resistant (MDR) isolates, but the overall VF score was higher for isolates of community and health care-associated origin than those of nosocomial origin (P = 0.0002 and P = 0.0172, respectively); the papA, papG allele II, hlyA, and hek VFs were more prevalent in this cohort. Most isolates belonged to phylogenetic group B2, which harbored a greater proportion of antibiotic-susceptible isolates than MDR isolates (P = 0.04). The community, health care-associated, or nosocomial origin of E. coli bacteremia determines the virulence capacity of an isolate better than the phylogenetic group does. This study provides new insights into the relationships between the pathogenesis and epidemiology of E. coli bacteremia.


Asunto(s)
Bacteriemia/microbiología , Infecciones Comunitarias Adquiridas/microbiología , Infección Hospitalaria/microbiología , Farmacorresistencia Bacteriana Múltiple , Infecciones por Escherichia coli/microbiología , Escherichia coli/aislamiento & purificación , Factores de Virulencia/genética , Adulto , Anciano , Anciano de 80 o más Años , Técnicas de Tipificación Bacteriana , Análisis por Conglomerados , Dermatoglifia del ADN , Escherichia coli/efectos de los fármacos , Escherichia coli/patogenicidad , Proteínas de Escherichia coli/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Epidemiología Molecular , Filogenia , Adulto Joven
16.
J Innate Immun ; 12(1): 90-102, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31266011

RESUMEN

Compared to females, males are more susceptible to acute viral and other respiratory tract infections that display greater severity and higher mortality. In contrast, females tend to fare worse with chronic inflammatory diseases. Circulating 17ß-estradiol (E2) is a female-specific factor that may influence the progression of human lung diseases. Here we hypothesize that E2 modulates the inflammatory response of monocytes through microRNA (miRNA)-based modulation of secretory leucoprotease inhibitor (SLPI), an antiprotease with immunomodulatory effects. Monocytic cells were treated ± E2, and differentially expressed miRNAs were identified using PCR profiling. Cells were transfected with miRNA mimics or antimiRs and SLPI mRNA and protein levels were quantified. Luciferase activity assay using wildtype and ΔmiR-19a/b-SLPI3'UTR reporter constructs and chromatin immunoprecipitation on E2-treated monocytes were performed. E2 downregulated SLPI and upregulated miR-19 expression in monocytes. Transfection with premiR-19b reduced SLPI mRNA and protein levels and this effect was abrogated using antimiRs against miR-19b. miR-19b directly binds the SLPI 3'UTR. The mechanism responsible for E2-mediated upregulation of miR-19 occurs via increased MIR17HG promoter activity mediated by c-MYC. Overall E2 decreases SLPI expression in human monocytic cells, via changes in miRNA expression and highlights the potential for estrogen to modulate the innate immune system.


Asunto(s)
MicroARNs/genética , Monocitos/metabolismo , Infecciones del Sistema Respiratorio/genética , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Factores Sexuales , Regiones no Traducidas 3'/genética , Células Cultivadas , Regulación hacia Abajo , Estradiol/metabolismo , Estrógenos/metabolismo , Femenino , Genes myc/genética , Humanos , Inmunidad Innata , Masculino , Regiones Promotoras Genéticas/genética , Infecciones del Sistema Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/genética , Caracteres Sexuales
17.
Pathogens ; 8(3)2019 06 28.
Artículo en Inglés | MEDLINE | ID: mdl-31261656

RESUMEN

Stenotrophomonas maltophilia is an emerging global opportunistic pathogen that has been appearing with increasing prevalence in cystic fibrosis (CF). A secreted protease from S. maltophilia has been reported as its chief potential virulence factor. Here, using the reference clinical strain S. maltophilia K279a, the major secreted proteases were identified. Protein biochemistry and mass spectrometry were carried out on K279a culture supernatant. The effect of K279a culture supernatant on cleavage and anti-neutrophil elastase activity of the three majors pulmonary antiproteases was quantified. A deletion mutant of S. maltophilia lacking expression of a protease was constructed. The serine proteases StmPR1, StmPR2 and StmPR3, in addition to chitinase A and an outer membrane esterase were identified in culture supernatants. Protease activity was incompletely abrogated in a K279a-ΔStmPR1: Erm mutant. Wild type K279a culture supernatant degraded alpha-1 antitrypsin (AAT), secretory leucoprotease inhibitor (SLPI) and elafin, important components of the lung's innate immune defences. Meanwhile SLPI and elafin, but not AAT, retained their ability to inhibit neutrophil elastase. StmPR3 together with StmPR1 and StmPR2, is likely to contribute to protease-mediated innate immune dysfunction in CF.

18.
Infect Immun ; 76(3): 1135-42, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18160475

RESUMEN

Escherichia coli is the principal gram-negative causative agent of sepsis and meningitis in neonates. The pathogenesis of meningitis due to E. coli K1 involves mucosal colonization, transcytosis of epithelial cells, survival in the bloodstream, and eventually invasion of the meninges. The last two aspects have been well characterized at a molecular level. Less is known about the early stages of pathogenesis, i.e., adhesion to and invasion of epithelial cells. We have previously reported that the Hek protein causes autoaggregation and can mediate adherence to and invasion of epithelial cells. Here, we report that Hek-mediated adherence is dependent on binding to glycosoaminoglycan, in particular, heparin. The ability to hemagglutinate, autoaggregate, adhere, and invade is contingent on a putative 25-amino-acid loop that is exposed to the outside of the bacterial cells.


Asunto(s)
Adhesinas de Escherichia coli/metabolismo , Adhesión Bacteriana , Células Epiteliales/microbiología , Escherichia coli/fisiología , Heparina/metabolismo , Adhesinas de Escherichia coli/química , Animales , Línea Celular , Sulfatos de Condroitina/metabolismo , Cricetinae , Sulfato de Dextran/metabolismo , Heparitina Sulfato/metabolismo , Modelos Moleculares , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Eliminación de Secuencia
19.
BMC Microbiol ; 8: 142, 2008 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-18778463

RESUMEN

BACKGROUND: The pagN gene of Salmonella enterica serovar Typhimurium is a PhoP-regulated gene that is up-regulated during growth within macrophages and in vivo in murine models of infection. The PagN protein displays similarity to the Hek and Tia invasins/adhesins of Escherichia coli. Thus far no function has been ascribed to the PagN protein. RESULTS: Here we show that the outer membrane located PagN protein mediates agglutination of red blood cells and that this can be masked by LPS. When expressed in Escherichia coli the PagN protein supports adhesion to and invasion of mammalian cells in a manner that is dependent on cytoskeletal rearrangements. S. enterica sv Typhimurium pagN mutants display a reduction in adhesion to and invasion of epithelial cells. Finally, we demonstrate that over-expression of PagN in a SPI-1 mutant can partially compensate for the lack of a functional invasasome. CONCLUSION: PagN is an outer membrane protein that may contribute to the virulence of S. Typhimurium. This protein is a haemagglutinin and contributes to the adherence to mammalian cells. In addition, PagN can mediate high-level invasion of CHO-K1 cells. Previously,pagN mutants have been shown to be less competitive in vivo and thus this may be due to their lessened ability to interact with mammalian cells. Finally PagN can be added to an ever-growing repertoire of factors that contribute to the pathogenesis of Salmonella.


Asunto(s)
Adhesinas Bacterianas/metabolismo , Salmonella typhimurium/metabolismo , Adhesinas Bacterianas/genética , Animales , Células CHO , Cricetinae , Cricetulus , Células HT29 , Hemaglutininas/metabolismo , Humanos , Mutación , Proteínas/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidad
20.
R Soc Open Sci ; 5(1): 171113, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-29410826

RESUMEN

Silver nanoparticle-based antimicrobials can promote a long lasting bactericidal effect without detrimental toxic side effects. However, there is not a clear and complete protocol to define and relate the properties of the particles (size, shape, surface charge, ionic content) with their specific activity. In this paper, we propose an effective multi-step approach for the identification of a 'purpose-specific active applicability window' to maximize the antimicrobial activity of medical devices containing silver nanoparticles (Ag NPs) (such as surface coaters), minimizing any consequent risk for human health (safety by design strategy). The antimicrobial activity and the cellular toxicity of four types of Ag NPs, differing in their coating composition and concentration have been quantified. Through the implementation of flow-field flow fractionation, Ag NPs have been characterized in terms of metal release, size and shape. The particles are fractionated in the process while being left unmodified, allowing for the identification of biological particle-specific contribution. Toxicity and inflammatory response in vitro have been assessed on human skin models, while antimicrobial activity has been monitored with both non-pathogenic and pathogenic Escherichia coli. The main benefit associated with such approach is the comprehensive assessment of the maximal effectiveness of candidate nanomaterials, while simultaneously indexing their properties against their safety.

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