RESUMEN
The ribosome is a large ribonucleoprotein assembly that uses diverse and complex molecular interactions to maintain proper folding. In vivo assembled ribosomes have been isolated using MS2 tags installed in either the 16S or 23S ribosomal RNAs (rRNAs), to enable studies of ribosome structure and function in vitro. RNA tags in the Escherichia coli 50S subunit have commonly been inserted into an extended helix H98 in 23S rRNA, as this addition does not affect cellular growth or in vitro ribosome activity. Here, we find that E. coli 50S subunits with MS2 tags inserted in H98 are destabilized compared to wild-type (WT) 50S subunits. We identify the loss of RNA-RNA tertiary contacts that bridge helices H1, H94, and H98 as the cause of destabilization. Using cryogenic electron microscopy (cryo-EM), we show that this interaction is disrupted by the addition of the MS2 tag and can be restored through the insertion of a single adenosine in the extended H98 helix. This work establishes ways to improve MS2 tags in the 50S subunit that maintain ribosome stability and investigates a complex RNA tertiary structure that may be important for stability in various bacterial ribosomes.
Asunto(s)
Escherichia coli , ARN Ribosómico , ARN Ribosómico/genética , ARN Ribosómico/análisis , Escherichia coli/genética , Ribosomas/genética , Ribosomas/química , ARN Ribosómico 23S/genética , ARN Ribosómico 23S/química , Subunidades Ribosómicas Grandes , ARN Bacteriano/genética , ARN Bacteriano/química , Proteínas RibosómicasRESUMEN
Type IVa pili are bacterial nanomachines required for colonization of surfaces, but little is known about the organization of proteins in this system. The Pseudomonas aeruginosa pilMNOPQ operon encodes five key members of the transenvelope complex facilitating pilus function. While PilQ forms the outer membrane secretin pore, the functions of the inner membrane-associated proteins PilM/N/O/P are less well defined. Structural characterization of a stable C-terminal fragment of PilP (PilP(Δ71)) by NMR revealed a modified ß-sandwich fold, similar to that of Neisseria meningitidis PilP, although complementation experiments showed that the two proteins are not interchangeable likely due to divergent surface properties. PilP is an inner membrane putative lipoprotein, but mutagenesis of the putative lipobox had no effect on the localization and function of PilP. A larger fragment, PilP(Δ18-6His), co-purified with a PilN(Δ44)/PilO(Δ51) heterodimer as a stable complex that eluted from a size exclusion chromatography column as a single peak with a molecular weight equivalent to two heterotrimers with 1:1:1 stoichiometry. Although PilO forms both homodimers and PilN-PilO heterodimers, PilP(Δ18-6His) did not interact stably with PilO(Δ51) alone. Together these data demonstrate that PilN/PilO/PilP interact directly to form a stable heterotrimeric complex, explaining the dispensability of PilP's lipid anchor for localization and function.
Asunto(s)
Proteínas Fimbrias/química , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Pseudomonas aeruginosa/metabolismo , Secuencia de Aminoácidos , Proteínas Fimbrias/genética , Fimbrias Bacterianas/química , Fimbrias Bacterianas/genética , Datos de Secuencia Molecular , Neisseria meningitidis/genética , Neisseria meningitidis/metabolismo , Operón , Unión Proteica , Estructura Terciaria de Proteína , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Alineación de SecuenciaRESUMEN
The highly conserved pilM/N/O/P/Q gene cluster is among the core set of genes required for cell surface expression of type IV pili and associated twitching motility. With the exception of the outer membrane secretin, a multimer of PilQ subunits, the specific functions of the products encoded by this gene cluster are poorly characterized. Orthologous proteins in the related bacterial type II secretion system have been shown to interact to form an inner membrane complex required for protein secretion. In this study, we provide evidence that the PilM/N/O/P proteins form a functionally equivalent type IVa pilus complex. Using Pseudomonas aeruginosa as model organism, we found that all four proteins, including the nominally cytoplasmic PilM, colocalized to the inner membrane. Stability studies via Western blot analyses revealed that loss of one component has a negative impact on the levels of other members of the putative complex. Furthermore, complementation studies revealed that the stoichiometry of the components is important for the correct formation of a stable complex in vivo. We provide evidence that an intact inner membrane complex is required for optimal formation of the outer membrane complex of the type IVa pilus system in P. aeruginosa, as PilQ stability is negatively affected in its absence. Finally, we show that, in the absence of the pilin subunit, the levels of membrane-bound components of the inner membrane complex are negatively regulated by the PilR/S two-component system, suggesting a role for PilR/S in sensing the piliation status of the cell.
Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Fimbrias Bacterianas/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas Bacterianas/genética , Proteínas Fimbrias/metabolismo , Genes Bacterianos , Prueba de Complementación Genética , Modelos Moleculares , Movimiento , Mutación/genética , Unión Proteica , Estabilidad Proteica , Transporte de Proteínas , Pseudomonas aeruginosa/genéticaRESUMEN
Type IV pili (T4P) are bacterial virulence factors responsible for attachment to surfaces and for twitching motility, a motion that involves a succession of pilus extension and retraction cycles. In the opportunistic pathogen Pseudomonas aeruginosa, the PilM/N/O/P proteins are essential for T4P biogenesis, and genetic and biochemical analyses strongly suggest that they form an inner-membrane complex. Here, we show through co-expression and biochemical analysis that the periplasmic domains of PilN and PilO interact to form a heterodimer. The structure of residues 69-201 of the periplasmic domain of PilO was determined to 2.2 A resolution and reveals the presence of a homodimer in the asymmetric unit. Each monomer consists of two N-terminal coiled coils and a C-terminal ferredoxin-like domain. This structure was used to generate homology models of PilN and the PilN/O heterodimer. Our structural analysis suggests that in vivo PilN/O heterodimerization would require changes in the orientation of the first N-terminal coiled coil, which leads to two alternative models for the role of the transmembrane domains in the PilN/O interaction. Analysis of PilN/O orthologues in the type II secretion system EpsL/M revealed significant similarities in their secondary structures and the tertiary structures of PilO and EpsM, although the way these proteins interact to form inner-membrane complexes appears to be different in T4P and type II secretion. Our analysis suggests that PilN interacts directly, via its N-terminal tail, with the cytoplasmic protein PilM. This work shows a direct interaction between the periplasmic domains of PilN and PilO, with PilO playing a key role in the proper folding of PilN. Our results suggest that PilN/O heterodimers form the foundation of the inner-membrane PilM/N/O/P complex, which is critical for the assembly of a functional T4P complex.
Asunto(s)
Proteínas Bacterianas/química , Periplasma/química , Multimerización de Proteína , Pseudomonas aeruginosa/química , Secuencia de Aminoácidos , Secuencia Conservada , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Homología Estructural de ProteínaRESUMEN
Growing skull fracture (GSF) is a progressive enlargement of a fracture due to an underlying tear of the dura mater. It is a rare complication of severe head injury mainly reported in young children. Classically, the diagnosis is made during follow-up, late after the original injury, when a palpable skull defect or a bulging mass is discovered clinically. Initial skull radiographs show a diastatic fracture developing later into a large bony defect. CT will show the brain damage which is usually present beneath the fracture. We present the MRI findings of GSF in a series of eight children. All patients initially had a large linear fracture and underlying brain damage on CT. In all cases MRI showed a zone of the same intensity as the brain contusion or cerebrospinal fluid advancing through the bone margins of the fracture to the subcutaneous plane. This finding was interpreted as an indirect sign of the dural tear. Seven patients were operated on with surgical confirmation of GSF. MRI can make an early diagnosis of GSF possible so that surgical repair with closure of the dura can be carried out before the dural tear enlarges.
Asunto(s)
Lesiones Encefálicas/diagnóstico , Imagen por Resonancia Magnética , Fracturas Craneales/diagnóstico , Encéfalo/patología , Niño , Preescolar , Duramadre/lesiones , Femenino , Humanos , Lactante , Masculino , Estudios Retrospectivos , Cráneo/patología , Factores de TiempoRESUMEN
The authors report their experience of imaging of 286 liver transplantations (LT) in children. Hepatic artery thrombosis is the most serious complication (9%), with a maximum risk during the first two weeks. Its clinical presentation is very variable and its diagnosis is based on Doppler ultrasonography. Emergency surgical disobstruction prevented the development of biliary or ischaemic complications in one half of cases. Portal thrombosis (4.5%) is due, in the majority of cases, to hypoplasia of the recipient's portal vein. Patency of intrahepatic portal branches with inversion of flow can delay the ultrasonographic diagnosis. Biliary complications are frequent (20%) and occur after a very variable interval. Their diagnosis is ultrasonographic, but sometimes delayed compared to the histological signs. Transhepatic cholangiography visualises the lesions and allows external drainage or even percutaneous dilatation. In 1 out of 4 cases, the aetiology was hepatic artery thrombosis. The imaging protocol after LT consists of ultrasonography with daily Doppler (or even twice-daily) in the patient's bed for the first two weeks and then as required. Computed tomography is useful to demonstrate parenchymal ischaemia and intraperitoneal abscess in multi-operated children. Angiography and cholangiography confirm the ultrasonographic signs. Interventional Radiology plays an increasingly important role.
Asunto(s)
Enfermedades de las Vías Biliares/etiología , Arteria Hepática/diagnóstico por imagen , Trasplante de Hígado/efectos adversos , Vena Porta/diagnóstico por imagen , Trombosis/etiología , Aortografía , Enfermedades de las Vías Biliares/diagnóstico por imagen , Niño , Preescolar , Colangiografía , Humanos , Complicaciones Posoperatorias , Trombosis/diagnóstico por imagen , Tomografía Computarizada por Rayos X , UltrasonografíaRESUMEN
Among a series of 140 liver transplantations in children, 21 biliary complications (BC) (15%) are reported. BC were identified from 2 days to 3 months after LT. Positive US findings were present in 20 cases. Cholangiography was obtained by opacification of a surgical drain in 3 cases, per-operatively in 3 and by PTC in 15. Drainage was placed in 9 and balloon dilatation performed in 2. Causes of BC include hepatic artery (HA) thrombosis in 7, HA stenosis in 1, anastomosis stricture in 7, anastomosis kink in 3, mucocele of cystic duct remnant in 2 and sludge in 1. Treatment was surgical in all, but 2 cases were treated percutaneously. There is a great difference in severity of prognosis between complications secondary to HA thrombosis and isolated BC. Role of US in diagnosis and of PTC and interventional radiology in treatment are emphasized.
Asunto(s)
Enfermedades de los Conductos Biliares/etiología , Trasplante de Hígado/efectos adversos , Adolescente , Enfermedades de los Conductos Biliares/diagnóstico , Enfermedades de los Conductos Biliares/diagnóstico por imagen , Enfermedades de los Conductos Biliares/terapia , Niño , Preescolar , Colangiografía , Humanos , Lactante , UltrasonografíaRESUMEN
The authors report the ultrasound and angiographic features of adenomas occurring in children with glycogen storage disease. Seven cases from 83 patients were diagnosed either by ultrasound, preoperative angiography or during surgery. The lesions appear on ultrasound as multiple rounded intrahepatic masses. Their degree of echogenicity as well of vascularity on angiography is highly variable. Ultrasound is the modality of choice in detecting adenomas. No malignant degeneration was observed.
Asunto(s)
Carcinoma Hepatocelular/complicaciones , Enfermedad del Almacenamiento de Glucógeno/complicaciones , Neoplasias Hepáticas/complicaciones , Adolescente , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/diagnóstico por imagen , Niño , Preescolar , Arteria Hepática/diagnóstico por imagen , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/diagnóstico por imagen , Radiografía , UltrasonografíaRESUMEN
The diagnosis of an acute hydrops of the gallbladder was done by ultrasonography during a mucocutaneous lymph node syndrome, with jaundice and hepatomegaly. Surgery was avoided when ultrasonographic follow-up showed the spontaneous resolution of the hydrops.