RESUMO
The ß-barrel assembly machinery (BAM complex) is essential for outer membrane protein (OMP) folding in Gram-negative bacteria, and represents a promising antimicrobial target. Several conformational states of BAM have been reported, but all have been obtained under conditions which lack the unique features and complexity of the outer membrane (OM). Here, we use Pulsed Electron-Electron Double Resonance (PELDOR, or DEER) spectroscopy distance measurements to interrogate the conformational ensemble of the BAM complex in E.â coli cells. We show that BAM adopts a broad ensemble of conformations in the OM, while in the presence of the antibiotic darobactin B (DAR-B), BAM's conformational equilibrium shifts to a restricted ensemble consistent with the lateral closed state. Our in-cell PELDOR findings are supported by new cryoEM structures of BAM in the presence and absence of DAR-B. This work demonstrates the utility of PELDOR to map conformational changes in BAM within its native cellular environment.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Dobramento de ProteínaRESUMO
The ß-barrel assembly machinery (BAM complex) is essential for outer membrane protein (OMP) folding in Gram-negative bacteria, and represents a promising antimicrobial target. Several conformational states of BAM have been reported, but all have been obtained under conditions which lack the unique features and complexity of the outer membrane (OM). Here, we use Pulsed Electron-Electron Double Resonance (PELDOR, or DEER) spectroscopy distance measurements to interrogate the conformational ensemble of the BAM complex in E.â coli cells. We show that BAM adopts a broad ensemble of conformations in the OM, while in the presence of the antibiotic darobactin B (DAR-B), BAM's conformational equilibrium shifts to a restricted ensemble consistent with the lateral closed state. Our in-cell PELDOR findings are supported by new cryoEM structures of BAM in the presence and absence of DAR-B. This work demonstrates the utility of PELDOR to map conformational changes in BAM within its native cellular environment.
RESUMO
INTRODUCTION: Nail guns are commonly used in the construction industry. They represent an occupational hazard, and in the context of mental illness can pose a threat to life. AIM: To determine the number of patients admitted to Auckland City Hospital (ACH) with a nail gun injury, and to review the current New Zealand legislation surrounding nail guns. METHODS: A 25-year retrospective review of patients admitted to ACH with a nail gun injury was performed by searching the ACH Trauma Registry. New Zealand legislation was reviewed. RESULTS: Between 1994 and 2019, 45 patients were admitted to ACH with a nail gun injury. Two subgroups were identified: 31% with an intentional injury; 69% with an unintentional injury. All patients were male. The mean age was 36.3. Patients with an intentional injury had a higher mortality rate (21.4% vs 9.5%), Injury Severity Scores (24.2 vs 3.4) and ICU admission rate (50% vs 3%) and required more intensive post-injury care when compared to unintentional injuries. There is currently no legislation in New Zealand specifically governing the use of nail guns. Only powder-actuated nail guns require certification. CONCLUSION: The continued occurrence of unintentional nail gun injuries and the high lethality of intentional injuries represent two distinct areas of concern. The Government should publish guidance aimed at improving safety and reducing the rate of intentional injury.
Assuntos
Indústria da Construção/instrumentação , Extremidades/lesões , Traumatismos Cranianos Penetrantes/epidemiologia , Traumatismos Ocupacionais/epidemiologia , Comportamento Autodestrutivo/epidemiologia , Ferimentos Penetrantes/epidemiologia , Adulto , Segurança de Equipamentos , Feminino , Traumatismos Cranianos Penetrantes/terapia , Hospitalização , Humanos , Escala de Gravidade do Ferimento , Unidades de Terapia Intensiva/estatística & dados numéricos , Tempo de Internação/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Mortalidade , Havaiano Nativo ou Outro Ilhéu do Pacífico , Nova Zelândia , Traumatismos Ocupacionais/terapia , Comportamento Autodestrutivo/terapia , Procedimentos Cirúrgicos Operatórios , População Branca , Ferimentos Penetrantes/terapiaRESUMO
The folding of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the ß-barrel assembly machinery (BAM). How lateral opening in the ß-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidrolases/metabolismo , Lipossomos/metabolismo , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Microscopia Crioeletrônica , Difusão Dinâmica da Luz , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/ultraestrutura , Hidrolases/genética , Hidrolases/isolamento & purificação , Hidrolases/ultraestrutura , Metabolismo dos Lipídeos , Lipossomos/ultraestrutura , Simulação de Dinâmica Molecular , Conformação Proteica em Folha beta , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestruturaRESUMO
BACKGROUND: Failure rates of up to 20% have been reported after fasciotomy for chronic exertional compartment syndrome (CECS). There is some evidence that postoperative failure and complication rates are higher in the posterior compartments of the lower leg than the anterolateral compartments. Isolated compartment surgery may put patients at risk of requiring revision surgery because of the risk of developing posterior compartment disease. HYPOTHESIS: Isolated anterolateral fasciotomy for CECS, in the absence of posterior compartment symptoms, produces satisfactory functional outcomes. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Between 2006 and 2012, patients who had positive intracompartment pressure-testing findings and who underwent isolated anterolateral fasciotomy release for CECS were given a self-administered questionnaire. The minimum follow-up was 3 years. The questionnaire addressed time to return to sport and ongoing symptoms. A visual analog scale was used to assess pain during exercise before and after surgery (score: 0, no pain; 10, worst pain imaginable); overall satisfaction with the procedure was assessed as well. Of 31 eligible patients, 20 patients (36 legs operated on) were assessed. RESULTS: Postoperatively, 90% of participants returned to the same or higher level of sport. The mean pain score during exercise before surgery was 8.17, whereas it was 1.74 after surgery. The overall mean patient satisfaction score was 8.64. Only 1 leg (2.8%) went on to develop posterior compartment syndrome. CONCLUSION: Isolated anterolateral fasciotomy for CECS produced excellent functional outcomes. Our rate of recurrence was low compared with those found in the literature, and 90% of participants returned to their same or higher level of sport postoperatively.
RESUMO
To determine if a high-fat diet (HF) from weaning would result in a pro-inflammatory state and affect joint cartilage, we fed male rats either HF or Chow diet post-weaning, and voluntary wheel exercise (EX) or cage only activity (SED) after 9 weeks of age. At 17 weeks body composition, plasma biomarkers and histomorphology scores of femoro-tibial cartilages of HF-SED, HF-EX, Chow-SED and Chow-EX groups were compared. Food intake and activity were not significantly different between groups. HF diet resulted in significantly higher weight gain, %fat, fat:lean ratio, and plasma leptin, insulin and TNFα concentrations, with significant interactions between diet and exercise. No abnormal features were detected in the hyaline articular cartilage or in the metaphyseal growth plate in all four groups. However, collagen type X- positive regions of retained epiphyseal growth cartilage (EGC) was present in all HF-fed animals and significantly greater than that observed in Chow-fed sedentary rats. Most lesions were located in the lateral posterior aspect of the tibia and/or femur. The severity of lesions was greater in HF-fed animals. Although exercise had a significantly greater effect in reducing adiposity and associated systemic inflammation in HF-fed rats, it had no effect on lesion incidence or severity. Lesion incidence was also significantly associated with indices of obesity and plasma markers of chronic inflammation. Clinically, EGC lesions induced by HF feeding in rats from very early in life, and possibly by insufficient activity, is typical of osteochondrosis in animals. Such lesions may be the precursor of juvenile osteochondritis dissecans requiring surgery in children/adolescents, conservative management of which could benefit from improved understanding of early changes in cellular and gene expression.