scholarly journals Refined Mechanism of Mycoplasma mobile Gliding Based on Structure, ATPase Activity, and Sialic Acid Binding of Machinery

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Miyuki S. Nishikawa ◽  
Daisuke Nakane ◽  
Takuma Toyonaga ◽  
Akihiro Kawamoto ◽  
Takayuki Kato ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Three Dimensional ◽  
Binding Activity ◽  
Influenza Viruses ◽  
Surface Proteins ◽  
Content Type ◽  
Distal Side ◽  
Atp Analogs ◽  
Electron Cryotomography

ABSTRACT Mycoplasma mobile, a fish pathogen, glides on solid surfaces by repeated catch, pull, and release of sialylated oligosaccharides by a unique mechanism based on ATP energy. The gliding machinery is composed of huge surface proteins and an internal “jellyfish”-like structure. Here, we elucidated the detailed three-dimensional structures of the machinery by electron cryotomography. The internal “tentacle”-like structure hydrolyzed ATP, which was consistent with the fact that the paralogs of the α- and β-subunits of F1-ATPase are at the tentacle structure. The electron microscopy suggested conformational changes of the tentacle structure depending on the presence of ATP analogs. The gliding machinery was isolated and showed that the binding activity to sialylated oligosaccharide was higher in the presence of ADP than in the presence of ATP. Based on these results, we proposed a model to explain the mechanism of M. mobile gliding. IMPORTANCE The genus Mycoplasma is made up of the smallest parasitic and sometimes commensal bacteria; Mycoplasma pneumoniae, which causes human “walking pneumonia,” is representative. More than ten Mycoplasma species glide on host tissues by novel mechanisms, always in the direction of the distal side of the machinery. Mycoplasma mobile, the fastest species in the genus, catches, pulls, and releases sialylated oligosaccharides (SOs), the carbohydrate molecules also targeted by influenza viruses, by means of a specific receptor and using ATP hydrolysis for energy. Here, the architecture of the gliding machinery was visualized three dimensionally by electron cryotomography (ECT), and changes in the structure and binding activity coupled to ATP hydrolysis were discovered. Based on the results, a refined mechanism was proposed for this unique motility.

scholarly journals Refined mechanism of Mycoplasma mobile gliding based on structure, ATPase activity, and sialic acid binding of machinery

2019 ◽  
Author(s):  
Miyuki S Nishikawa ◽  
Daisuke Nakane ◽  
Takuma Toyonaga ◽  
Akihiro Kawamoto ◽  
Takayuki Kato ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Three Dimensional ◽  
Binding Activity ◽  
Influenza Viruses ◽  
Surface Proteins ◽  
Solid Surfaces ◽  
Distal Side ◽  
Atp Analogs ◽  
Electron Cryotomography

ABSTRACTMycoplasma mobile, a fish pathogen, glides on solid surfaces by repeated catch, pull, and release of sialylated oligosaccharides by a unique mechanism based on ATP energy. The gliding machinery is composed of huge surface proteins and an internal jellyfish -like structure. Here, we elucidated the detailed three-dimensional structures of the machinery by electron cryotomography. The internal tentacle -like structure hydrolyzed ATP, which was consistent with the fact that the paralogs of the α- and β-subunits of F1-ATPase are at the tentacle structure. The electron microscopy suggested conformational changes of the tentacle structure depending on the presence of ATP analogs. The gliding machinery was isolated and shown that the binding activity to sialylated oligosaccharide was higher in the presence of ADP than in the presence of ATP. Based on these results, we proposed a model to explain the mechanism of M. mobile gliding.IMPORTANCEThe genus Mycoplasma is made up of the smallest parasitic and sometimes commensal bacteria; Mycoplasma pneumoniae, which causes human walking pneumonia, is representative. More than ten Mycoplasma species glide on host tissues by novel mechanisms always in the direction of the distal side of the machinery. Mycoplasma mobile, the fastest species in the genus, catches, pulls, and releases sialylated oligosaccharides (SOs), the carbohydrate molecules also targeted by influenza viruses, by means of a specific receptor and using ATP hydrolysis for energy. Here, the architecture of the gliding machinery was visualized three-dimensionally by electron cryotomography (ECT), and changes in the structure and binding activity coupled to ATP hydrolysis were discovered. Based on the results, a refined mechanism was proposed for this unique motility.

scholarly journals Periodicity in Attachment Organelle Revealed by Electron Cryotomography Suggests Conformational Changes in Gliding Mechanism ofMycoplasma pneumoniae

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Akihiro Kawamoto ◽  
Lisa Matsuo ◽  
Takayuki Kato ◽  
Hiroki Yamamoto ◽  
Keiichi Namba ◽  
...  
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Conformational Changes ◽  
Hexagonal Lattice ◽  
Gliding Motility ◽  
Influenza Viruses ◽  
Pathogenic Bacterium ◽  
Dimensional Structure ◽  
Internal Core ◽  
Electron Cryotomography ◽  
Gliding Mechanism

ABSTRACTMycoplasma pneumoniae, a pathogenic bacterium, glides on host surfaces using a unique mechanism. It forms an attachment organelle at a cell pole as a protrusion comprised of knoblike surface structures and an internal core. Here, we analyzed the three-dimensional structure of the organelle in detail by electron cryotomography. On the surface, knoblike particles formed a two-dimensional array, albeit with limited regularity. Analyses using a nonbinding mutant and an antibody showed that the knoblike particles correspond to a naplike structure that has been observed by negative-staining electron microscopy and is likely to be formed as a complex of P1 adhesin, the key protein for binding and gliding. The paired thin and thick plates feature a rigid hexagonal lattice and striations with highly variable repeat distances, respectively. The combination of variable and invariant structures in the internal core and the P1 adhesin array on the surface suggest a model in which axial extension and compression of the thick plate along a rigid thin plate is coupled with attachment to and detachment from the substrate during gliding.IMPORTANCEHuman mycoplasma pneumonia, epidemic all over the world in recent years, is caused by a pathogenic bacterium,Mycoplasma pneumoniae. This tiny bacterium, about 2 µm in cell body length, glides on the surface of the human trachea to infect the host by binding to sialylated oligosaccharides, which are also the binding targets of influenza viruses. The mechanism of mycoplasmal gliding motility is not related to any other well-studied motility systems, such as bacterial flagella and cytoplasmic motor proteins. Here, we visualized the attachment organelle, a cellular architecture for gliding, three dimensionally by using electron cryotomography and other conventional methods. A possible gliding mechanism has been suggested based on the architectural images.

scholarly journals Mutations on the N-Terminal Edge of the DELSEED Loop in either the α or β Subunit of the Mitochondrial F 1 -ATPase Enhance ATP Hydrolysis in the Absence of the Central γ Rotor

2013 ◽  
Vol 12 (11) ◽  
pp. 1451-1461 ◽  
Author(s):  
Thuy La ◽  
George Desmond Clark-Walker ◽  
Xiaowen Wang ◽  
Stephan Wilkens ◽  
Xin Jie Chen
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Kluyveromyces Lactis ◽  
Β Subunit ◽  
Α Subunit ◽  
Content Type ◽  
Subunit Stoichiometry ◽  
Catalytic Sites ◽  
A Subunit

ABSTRACT F 1 -ATPase is a rotary molecular machine with a subunit stoichiometry of α 3 β 3 γ 1 δ 1 ε 1 . It has a robust ATP-hydrolyzing activity due to effective cooperativity between the three catalytic sites. It is believed that the central γ rotor dictates the sequential conformational changes to the catalytic sites in the α 3 β 3 core to achieve cooperativity. However, recent studies of the thermophilic Bacillus PS3 F 1 -ATPase have suggested that the α 3 β 3 core can intrinsically undergo unidirectional cooperative catalysis (T. Uchihashi et al., Science 333:755-758, 2011). The mechanism of this γ-independent ATP-hydrolyzing mode is unclear. Here, a unique genetic screen allowed us to identify specific mutations in the α and β subunits that stimulate ATP hydrolysis by the mitochondrial F 1 -ATPase in the absence of γ. We found that the F446I mutation in the α subunit and G419D mutation in the β subunit suppress cell death by the loss of mitochondrial DNA (ρ o ) in a Kluyveromyces lactis mutant lacking γ. In organello ATPase assays showed that the mutant but not the wild-type γ-less F 1 complexes retained 21.7 to 44.6% of the native F 1 -ATPase activity. The γ-less F 1 subcomplex was assembled but was structurally and functionally labile in vitro . Phe446 in the α subunit and Gly419 in the β subunit are located on the N-terminal edge of the DELSEED loops in both subunits. Mutations in these two sites likely enhance the transmission of catalytically required conformational changes to an adjacent α or β subunit, thereby allowing robust ATP hydrolysis and cell survival under ρ o conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the α 3 β 3 subcomplex.

scholarly journals A Nonfimbrial Adhesin ofAggregatibacter actinomycetemcomitansMediates Biofilm Biogenesis

2018 ◽  
Vol 87 (1) ◽  
Author(s):  
David R. Danforth ◽  
Gaoyan Tang-Siegel ◽  
Teresa Ruiz ◽  
Keith P. Mintz
Keyword(s):  
Biofilm Formation ◽  
Protein A ◽  
Three Dimensional ◽  
Binding Activity ◽  
Periodontal Pathogen ◽  
Collagen Binding ◽  
Content Type ◽  
Mutant Strains ◽  
Different Strains ◽  
Biofilm Development

ABSTRACTPeriodontitis is an inflammatory disease caused by polymicrobial biofilms. The periodontal pathogenAggregatibacter actinomycetemcomitansdisplays two proteinaceous surface structures, the fimbriae and the nonfimbrial extracellular matrix binding protein A (EmaA), as observed by electron microscopy. Fimbriae participate in biofilm biogenesis and the EmaA adhesins mediate collagen binding. However, in the absence of fimbriae,A. actinomycetemcomitansstill retains the potential to form robust biofilms, suggesting that other surface macromolecules participate in biofilm development. Here, isogenic mutant strains lacking EmaA structures, but still expressing fimbriae, were observed to have reduced biofilm potential. In strains lacking both EmaA and fimbriae, biofilm mass was reduced by 80%. EmaA enhanced biofilm formation in different strains, independent of the fimbriation state or serotype. Confocal microscopy revealed differences in cell density within microcolonies between the EmaA positive and mutant strains. EmaA-mediated biofilm formation was found to be independent of the glycosylation state and the precise three-dimensional conformation of the protein, and thus this function is uncorrelated with collagen binding activity. The data suggest that EmaA is a multifunctional adhesin that utilizes different mechanisms to enhance bacterial binding to collagen and to enhance biofilm formation, both of which are important forA. actinomycetemcomitanscolonization and subsequent infection.

Effects of arterial geometry on aneurysm growth: three-dimensional computational fluid dynamics study

2004 ◽  
Vol 101 (4) ◽  
pp. 676-681 ◽  
Author(s):  
Yiemeng Hoi ◽  
Hui Meng ◽  
Scott H. Woodward ◽  
Bernard R. Bendok ◽  
Ricardo A. Hanel ◽  
...  
Keyword(s):  
Fluid Dynamics ◽  
Computational Fluid Dynamics ◽  
Three Dimensional ◽  
Impact Zone ◽  
Content Type ◽  
Aneurysm Neck ◽  
Distal Side ◽  
Aneurysm Growth ◽  
Fine Print ◽  
Saccular Aneurysms

Object. Few researchers have quantified the role of arterial geometry in the pathogenesis of saccular cerebral aneurysms. The authors investigated the effects of parent artery geometry on aneurysm hemodynamics and assessed the implications relative to aneurysm growth and treatment effectiveness. Methods. The hemodynamics of three-dimensional saccular aneurysms arising from the lateral wall of arteries with varying arterial curves (starting with a straight vessel model) and neck sizes were studied using a computational fluid dynamics analysis. The effects of these geometric parameters on hemodynamic parameters, including flow velocity, aneurysm wall shear stress (WSS), and area of elevated WSS during the cardiac cycle (time-dependent impact zone), were quantified. Unlike simulations involving aneurysms located on straight arteries, blood flow inertia (centrifugal effects) rather than viscous diffusion was the predominant force driving blood into aneurysm sacs on curved arteries. As the degree of arterial curvature increased, flow impingement on the distal side of the neck intensified, leading to elevations in the WSS and enlargement of the impact zone at the distal side of the aneurysm neck. Conclusions. Based on these simulations the authors postulate that lateral saccular aneurysms located on more curved arteries are subjected to higher hemodynamic stresses. Saccular aneurysms with wider necks have larger impact zones. The large impact zone at the distal side of the aneurysm neck correlates well with other findings, implicating this zone as the most likely site of aneurysm growth or regrowth of treated lesions. To protect against high hemodynamic stresses, protection of the distal side of the aneurysm neck from flow impingement is critical.

scholarly journals Cross-linking of bovine rhodopsin with sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate affects its functionality

2020 ◽  
Vol 477 (12) ◽  
pp. 2295-2312
Author(s):  
Rafael Medina ◽  
Deisy Perdomo ◽  
Carolina Möller ◽  
José Bubis
Keyword(s):  
Conformational Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Three Dimensional ◽  
Binding Activity ◽  
Dimensional Structure ◽  
Cross Linking ◽  
Bovine Rhodopsin ◽  
Rhodopsin Kinase ◽  
Cross Links ◽  
Guanine Nucleotide Binding

Rhodopsin is the photoreceptor protein involved in visual excitation in retinal rods. The functionality of bovine rhodopsin was determined following treatment with sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), a bifunctional reagent capable of forming covalent cross-links between suitable placed lysines and cysteines. Denaturing polyacrylamide gel electrophoresis showed that rhodopsin incubated with sulfo-SMCC generated intermolecular dimers, trimers, and higher oligomers, although most of the sulfo-SMCC-treated protein remained as a monomer. Minor alterations on the absorption spectrum of light-activated sulfo-SMCC-treated rhodopsin were observed. However, only ∼2% stimulation of the guanine nucleotide binding activity of transducin was measured in the presence of sulfo-SMCC-cross-linked photolyzed rhodopsin. Moreover, rhodopsin kinase was not able of phosphorylating sulfo-SMCC-cross-linked rhodopsin after illumination. Rhodopsin was purified in the presence of either 0.1% or 1% n-dodecyl β-d-maltoside, to obtain dimeric and monomeric forms of the protein, respectively. Interestingly, no generation of the regular F1 and F2 thermolytic fragments was perceived with sulfo-SMCC-cross-linked rhodopsin either in the dimeric or monomeric state, implying the formation of intramolecular connections in the protein that might thwart the light-induced conformational changes required for interaction with transducin and rhodopsin kinase. Structural analysis of the rhodopsin three-dimensional structure suggested that the following lysine and cysteine pairs: Lys66/Lys67 and Cys316, Cys140 and Lys141, Cys140 and Lys248, Lys311 and Cys316, and/or Cys316 and Lys325 are potential candidates to generate intramolecular cross-links in the protein. Yet, the lack of fragmentation of sulfo-SMCC-treated Rho with thermolysin is consistent with the formation of cross-linking bridges between Lys66/Lys67 and Cys316, and/or Cys140 and Lys248.

scholarly journals Honey Bee Deformed Wing Virus Structures Reveal that Conformational Changes Accompany Genome Release

2016 ◽  
Vol 91 (2) ◽  
Author(s):  
Lindsey J. Organtini ◽  
Kristin L. Shingler ◽  
Robert E. Ashley ◽  
Elizabeth A. Capaldi ◽  
Kulsoom Durrani ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Honey Bees ◽  
Three Dimensional ◽  
Open Flower ◽  
Deformed Wing Virus ◽  
Content Type ◽  
Empty Capsid ◽  
Infected Adult ◽  
Colony Collapse ◽  
Global Agriculture

ABSTRACT The picornavirus-like deformed wing virus (DWV) has been directly linked to colony collapse; however, little is known about the mechanisms of host attachment or entry for DWV or its molecular and structural details. Here we report the three-dimensional (3-D) structures of DWV capsids isolated from infected honey bees, including the immature procapsid, the genome-filled virion, the putative entry intermediate (A-particle), and the empty capsid that remains after genome release. The capsids are decorated by large spikes around the 5-fold vertices. The 5-fold spikes had an open flower-like conformation for the procapsid and genome-filled capsids, whereas the putative A-particle and empty capsids that had released the genome had a closed tube-like spike conformation. Between the two conformations, the spikes undergo a significant hinge-like movement that we predicted using a Robetta model of the structure comprising the spike. We conclude that the spike structures likely serve a function during host entry, changing conformation to release the genome, and that the genome may escape from a 5-fold vertex to initiate infection. Finally, the structures illustrate that, similarly to picornaviruses, DWV forms alternate particle conformations implicated in assembly, host attachment, and RNA release. IMPORTANCE Honey bees are critical for global agriculture, but dramatic losses of entire hives have been reported in numerous countries since 2006. Deformed wing virus (DWV) and infestation with the ectoparasitic mite Varroa destructor have been linked to colony collapse disorder. DWV was purified from infected adult worker bees to pursue biochemical and structural studies that allowed the first glimpse into the conformational changes that may be required during transmission and genome release for DWV.

scholarly journals Fhb, a Novel Factor H-Binding Surface Protein, Contributes to the Antiphagocytic Ability and Virulence of Streptococcus suis

2012 ◽  
Vol 80 (7) ◽  
pp. 2402-2413 ◽  
Author(s):  
Yaya Pian ◽  
Shuzhen Gan ◽  
Shujie Wang ◽  
Jie Guo ◽  
Pingping Wang ◽  
...  
Keyword(s):  
Binding Protein ◽  
Surface Protein ◽  
Streptococcus Suis ◽  
Invasive Disease ◽  
Binding Activity ◽  
Repeat Sequence ◽  
Factor H ◽  
Surface Proteins ◽  
Infection Model ◽  
Content Type

ABSTRACTStreptococcus suisserotype 2 is a Gram-positive bacterium that causes sepsis and meningitis in piglets and humans. The mechanisms ofS. suisserotype 2 invasive disease are not well understood. The surface proteins of pathogens usually play important roles in infection and bacterium-host interactions. Here, we identified a novel surface protein that contributed significantly to the virulence ofS. suisserotype 2 in a piglet infection model. This protein showed little similarity to other reported proteins and exhibited strong binding activity to human factor H (hFH). It was designated Fhb (factor H-binding protein). Thefhbgenes found inS. suisserotypes 1, 2, 4, 7, and 9 exhibited molecular polymorphism. Fhb possessed two proline-rich repeat sequences and XPZ domains, and one repeat sequence exhibited a high homology to Bac, an IgA-binding protein ofStreptococcus agalactiae. Evidence strongly indicated thatfhb-deficient mutants had diminished phagocytosis resistance in bactericidal assays. In addition, Fhb plays important roles in complement-mediated immunity by interacting with hFH. These findings indicated that Fhb is a crucial surface protein contributing to the virulence ofS. suis, with important functions in evading innate immune defenses by interaction with host complement regulatory factor hFH.

scholarly journals Minicells, Back in Fashion

2016 ◽  
Vol 198 (8) ◽  
pp. 1186-1195 ◽  
Author(s):  
Madeline M. Farley ◽  
Bo Hu ◽  
William Margolin ◽  
Jun Liu
Keyword(s):  
Conformational Changes ◽  
Electron Tomography ◽  
Imaging Techniques ◽  
Three Dimensional ◽  
Model Systems ◽  
Specimen Thickness ◽  
Macromolecular Complexes ◽  
Content Type ◽  
Cellular Environment

Cryo-electron tomography (cryo-ET) has emerged as a leading technique for three-dimensional visualization of large macromolecular complexes and their conformational changes in their native cellular environment. However, the resolution and potential applications of cryo-ET are fundamentally limited by specimen thickness, preventing high-resolutionin situvisualization of macromolecular structures in many bacteria (such asEscherichia coliandSalmonella enterica). Minicells, which were discovered nearly 50 years ago, have recently been exploited as model systems to visualize molecular machinesin situ, due to their smaller size and other unique properties. In this review, we discuss strategies for producing minicells and highlight their use in the study of chemotactic signaling, protein secretion, and DNA translocation. In combination with powerful genetic tools and advanced imaging techniques, minicells provide a springboard for in-depth structural studies of bacterial macromolecular complexesin situand therefore offer a unique approach for gaining novel structural insights into many important processes in microbiology.

Electron Microscopy and image reconstruction reveal the structural basis for spectrin's elastic properties

1992 ◽  
Vol 50 (1) ◽  
pp. 510-511
Author(s):  
Amy M. McGough ◽  
Robert Josephs
Keyword(s):  
Electron Microscopy ◽  
Elastic Properties ◽  
Conformational Changes ◽  
Quaternary Structure ◽  
Three Dimensional ◽  
Membrane Skeleton ◽  
Projection Algorithm ◽  
Structural Basis ◽  
Iterative Approximation ◽  
Membrane Skeletons

The remarkable deformability of the erythrocyte derives in large part from the elastic properties of spectrin, the major component of the membrane skeleton. It is generally accepted that spectrin's elasticity arises from marked conformational changes which include variations in its overall length (1). In this work the structure of spectrin in partially expanded membrane skeletons was studied by electron microscopy to determine the molecular basis for spectrin's elastic properties. Spectrin molecules were analysed with respect to three features: length, conformation, and quaternary structure. The results of these studies lead to a model of how spectrin mediates the elastic deformation of the erythrocyte.Membrane skeletons were isolated from erythrocyte membrane ghosts, negatively stained, and examined by transmission electron microscopy (2). Particle lengths and end-to-end distances were measured from enlarged prints using the computer program MACMEASURE. Spectrin conformation (straightness) was assessed by calculating the particles’ correlation length by iterative approximation (3). Digitised spectrin images were correlation averaged or Fourier filtered to improve their signal-to-noise ratios. Three-dimensional reconstructions were performed using a suite of programs which were based on the filtered back-projection algorithm and executed on a cluster of Microvax 3200 workstations (4).

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