Unitary step of gliding machinery in Mycoplasma mobile.
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ABSTRACT:
SUBMITTER: Kinosita Y
PROVIDER: S-EPMC4060671 | biostudies-other | 2014 Jun
REPOSITORIES: biostudies-other
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Project description:Mycoplasma mobile, a fish pathogen, exhibits its own specialized gliding motility on host cells based on ATP hydrolysis. The special protein machinery enabling this motility is composed of surface and internal protein complexes. Four proteins, MMOBs 1630, 1660, 1670, and 4860 constitute the internal complex, including paralogs of F-type ATPase/synthase ? and ? subunits. In the present study, the cellular localisation for the candidate gliding machinery proteins, MMOBs 1620, 1640, 1650, and 5430 was investigated by using a total internal reflection fluorescence microscopy system after tagging these proteins with the enhanced yellow fluorescent protein (EYFP). The M. mobile strain expressing a fusion protein MMOB1620-EYFP exhibited reduced cell-binding activity and a strain expressing MMOB1640 fused with EYFP exhibited increased gliding speed, showing the involvement of these proteins in the gliding mechanism. Based on the genomic sequences, we analysed the sequence conservativity in the proteins of the internal and the surface complexes from four gliding mycoplasma species. The proteins in the internal complex were more conserved compared to the surface complex, suggesting that the surface complex undergoes modifications depending on the host. The analyses suggested that the internal gliding complex was highly conserved probably due to its role in the motility mechanism.
| S-EPMC7052211 | biostudies-literature
Project description:UNLABELLED:Mycoplasma mobile glides in the direction of its cell pole by a unique mechanism in which hundreds of legs, each protruding from its own gliding unit, catch, pull, and release sialylated oligosaccharides fixed on a solid surface. In this study, we found that 77% of cells glided to the left with a change in direction of 8.4° ± 17.6° ?m(-1) displacement. The cell body did not roll around the cell axis, and elongated, thinner cells also glided while tracing a curved trajectory to the left. Under viscous conditions, the range of deviation of the gliding direction decreased. In the presence of 250 ?M free sialyllactose, in which the binding of the legs (i.e., the catching of sialylated oligosaccharides) was reduced, 70% and 30% of cells glided to the left and the right, respectively, with changes in direction of ?30° ?m(-1). The gliding ghosts, in which a cell was permeabilized by Triton X-100 and reactivated by ATP, glided more straightly. These results can be explained by the following assumptions based on the suggested gliding machinery and mechanism: (i) the units of gliding machinery may be aligned helically around the cell, (ii) the legs extend via the process of thermal fluctuation and catch the sialylated oligosaccharides, and (iii) the legs generate a propulsion force that is tilted from the cell axis to the left in 70% and to the right in 30% of cells. IMPORTANCE:Mycoplasmas are bacteria that are generally parasitic to animals and plants. Some Mycoplasma species form a protrusion at a pole, bind to solid surfaces, and glide. Although these species appear to consistently glide in the direction of the protrusion, their exact gliding direction has not been examined. This study analyzed the gliding direction in detail under various conditions and, based on the results, suggested features of the machinery and the mechanism of gliding.
| S-EPMC4751794 | biostudies-literature
Project description:Several species of mycoplasmas glide on solid surfaces, in the direction of their membrane protrusion at a cell pole, by an unknown mechanism. Our recent studies on the fastest species, Mycoplasma mobile, suggested that the gliding machinery, localized at the base of the membrane protrusion (the "neck"), is composed of two huge proteins. This machinery forms spikes sticking out from the neck and propels the cell by alternately binding and unbinding the spikes to a solid surface. Here, to study the intracellular mechanisms for gliding, we established a permeabilized gliding ghost model, analogous to the "Triton model" of the eukaryotic axoneme. Treatment with Triton X-100 stopped the gliding and converted the cells to permeabilized "ghosts." When ATP was added exogenously, approximately 85% of the ghosts were reactivated, gliding at speeds similar to those of living cells. The reactivation activity and inhibition by various nucleotides and ATP analogs, as well as their kinetic parameters, showed that the machinery is driven by the hydrolysis of ATP to ADP plus phosphate, caused by an unknown ATPase.
| S-EPMC1192825 | biostudies-literature
Project description:Mycoplasma mobile, a fish pathogen, exhibits gliding motility using ATP hydrolysis on solid surfaces, including animal cells. The gliding machinery can be divided into surface and internal structures. The internal structure of the motor is composed of 28 so-called "chains" that are each composed of 17 repeating protein units called "particles." These proteins include homologs of the catalytic α and β subunits of F1-ATPase. In this study, we isolated the particles and determined their structures using negative-staining electron microscopy and high-speed atomic force microscopy. The isolated particles were composed of five proteins, MMOB1660 (α-subunit homolog), -1670 (β-subunit homolog), -1630, -1620, and -4530, and showed ATP hydrolyzing activity. The two-dimensional (2D) structure, with dimensions of 35 and 26 nm, showed a dimer of hexameric ring approximately 12 nm in diameter, resembling F1-ATPase catalytic (αβ)3. We isolated the F1-like ATPase unit, which is composed of MMOB1660, -1670, and -1630. Furthermore, we isolated the chain and analyzed the three-dimensional (3D) structure, showing that dimers of mushroom-like structures resembling F1-ATPase were connected and aligned along the dimer axis at 31-nm intervals. An atomic model of F1-ATPase catalytic (αβ)3 from Bacillus PS3 was successfully fitted to each hexameric ring of the mushroom-like structure. These results suggest that the motor for M. mobile gliding shares an evolutionary origin with F1-ATPase. Based on the obtained structure, we propose possible force transmission processes in the gliding mechanism. IMPORTANCE F1Fo-ATPase, a rotary ATPase, is widespread in the membranes of mitochondria, chloroplasts, and bacteria and converts ATP energy with a proton motive force across the membrane by its physical rotation. Homologous protein complexes play roles in ion and protein transport. Mycoplasma mobile, a pathogenic bacterium, was recently suggested to have a special motility system evolutionarily derived from F1-ATPase. The present study isolated the protein complex from Mycoplasma cells and supported this conclusion by clarifying the detailed structures containing common and novel features as F1-ATPase relatives.
| S-EPMC8406192 | biostudies-literature
Project description: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.
| S-EPMC6935860 | biostudies-literature
Project description:UnlabelledThe binding and gliding of Mycoplasma mobile on a plastic plate covered by 53 uniform oligosaccharides were analyzed. Mycoplasmas bound to and glided on only 21 of the fixed sialylated oligosaccharides (SOs), showing that sialic acid is essential as the binding target. The affinities were mostly consistent with our previous results on the inhibitory effects of free SOs and suggested that M. mobile recognizes SOs from the nonreducing end with four continuous sites as follows. (i and ii) A sialic acid at the nonreducing end is tightly recognized by tandemly connected two sites. (iii) The third site is recognized by a loose groove that may be affected by branches. (iv) The fourth site is recognized by a large groove that may be enhanced by branches, especially those with a negative charge. The cells glided on uniform SOs in manners apparently similar to those of the gliding on mixed SOs. The gliding speed was related inversely to the mycoplasma's affinity for SO, suggesting that the detaching step may be one of the speed determinants. The cells glided faster and with smaller fluctuations on the uniform SOs than on the mixtures, suggesting that the drag caused by the variation in SOs influences gliding behaviors.ImportanceMycoplasma is a group of bacteria generally parasitic to animals and plants. Some Mycoplasma species form a protrusion at a pole, bind to solid surfaces, and glide in the direction of the protrusion. These procedures are essential for parasitism. Usually, mycoplasmas glide on mixed sialylated oligosaccharides (SOs) derived from glycoprotein and glycolipid. Since gliding motility on uniform oligosaccharides has never been observed, this study gives critical information about recognition and interaction between receptors and SOs.
| S-EPMC4542179 | biostudies-literature
Project description:Mycoplasma mobile is a bacterium that uses a unique mechanism to glide on solid surfaces at a velocity of up to 4.5 ?m/s. Its gliding machinery comprises hundreds of units that generate the force for gliding based on the energy derived from ATP; the units catch and pull sialylated oligosaccharides fixed to solid surfaces. In this study, we measured the stall force of wild-type and mutant strains of M. mobile carrying a bead manipulated using optical tweezers. The strains that had been enhanced for binding exhibited weaker stall forces than the wild-type strain, indicating that stall force is related to force generation rather than to binding. The stall force of the wild-type strain decreased linearly from 113 to 19 picoNewtons after the addition of 0-0.5 mM free sialyllactose (a sialylated oligosaccharide), with a decrease in the number of working units. After the addition of 0.5 mM sialyllactose, the cells carrying a bead loaded using optical tweezers exhibited stepwise movements with force increments. The force increments ranged from 1 to 2 picoNewtons. Considering the 70-nm step size, this small-unit force may be explained by the large gear ratio involved in the M. mobile gliding machinery.
| S-EPMC5883615 | biostudies-literature
Project description:Mycoplasma mobile is a fish pathogen that glides on solid surfaces by means of its own gliding machinery composed of internal and surface structures. In the present study, we focused on the function and structure of Gli123, a surface protein that is essential for the localization of other surface proteins. The amino acid sequence of Gli123, which is 1,128 amino acids long, contains lipoprotein-specific repeats. We isolated the native Gli123 protein from M. mobile cells and a recombinant protein, rGli123, from Escherichia coli. The isolated rGli123 complemented a nonbinding and nongliding mutant of M. mobile that lacked Gli123. Circular dichroism and rotary-shadowing electron microscopy (EM) showed that rGli123 has a structure that is not significantly different from that of the native protein. Rotary-shadowing EM suggested that Gli123 adopts two distinct globular and rod-like structures, depending on the ionic strength of the solution. Negative-staining EM coupled with single-particle analysis revealed that Gli123 forms a globular structure featuring a small protrusion with dimensions of approximately 15.7, 14.7, and 14.1 nm for the "height," major axis and minor axis, respectively. Small-angle X-ray scattering analyses indicated a rod-like structure composed of several tandem globular domains with total dimensions of approximately 34 nm in length and 6 nm in width. Both molecular structures were suggested to be dimers, based on the predicted molecular size and structure. Gli123 may have evolved by multiplication of repeating lipoprotein units and acquired a role for Gli521 and Gli349 assembly. IMPORTANCE Mycoplasmas are pathogenic bacteria that are widespread in animals. They are characterized by small cell and genome sizes but are equipped with unique abilities for infection, such as surface variation and gliding. Here, we focused on a surface-localizing protein named Gli123 that is essential for Mycoplasma mobile gliding. This study suggested that Gli123 undergoes drastic conformational changes between its rod-like and globular structures. These changes may be caused by a repetitive structure common in the surface proteins that is responsible for the modulation of the cell surface structure and related to the assembly process for the surface gliding machinery. An evolutionary process for surface proteins essential for this mycoplasma gliding was also suggested in the present study.
| S-EPMC10029712 | biostudies-literature
Project description:Mycoplasma mobile glides on solid surfaces by the repeated binding of leg structures to sialylated oligosaccharide fixed on a solid surface. To obtain information about the propulsion caused by the leg, we made elongated and stiff cells using a detergent. Within 30 min after the cells were treated with 0.1% Tween 60, the cells were elongated from 0.8 ?m to 2.2 ?m in length while maintaining their gliding activity. Fluorescence and electron microscopy showed that a part of the cytoskeletal structure was elongated, while the localization of proteins involved in the gliding was not modified significantly. The elongated cells glided with repeated pivoting around the cellular position of gliding machinery by 10 degrees of amplitude at a frequency of 2 to 3 times per second, suggesting that the propulsion in a line perpendicular to the cell axis can occur with different timings. The pivoting speed decreased as the cell length increased, probably from the load generated by the friction. The torque required to achieve the actual pivoting increased with the cell length without saturation, reaching 54.7 pN nm at 4.3 ?m in cell length.
| S-EPMC3256606 | biostudies-literature
Project description:Mycoplasmas, which have been shown to be the causative pathogens in recent human pneumonia epidemics, bind to solid surfaces and glide in the direction of the membrane protrusion at a pole. During gliding, the legs of the mycoplasma catch, pull, and release sialylated oligosaccharides fixed on a solid surface. Sialylated oligosaccharides are major structures on animal cell surfaces and are sometimes targeted by pathogens, such as influenza virus. In the present study, we analyzed the inhibitory effects of 16 chemically synthesized sialylated compounds on the gliding and binding of Mycoplasma mobile and Mycoplasma pneumoniae and concluded the following. (i) The recognition of sialylated oligosaccharide by mycoplasma legs proceeds in a "lock-and-key" fashion, with the binding affinity dependent on structural differences among the sialylated compounds examined. (ii) The binding of the leg and the sialylated oligosaccharide is cooperative, with Hill constants ranging from 2 to 3. (iii) Mycoplasma legs may generate a drag force after a stroke, because the gliding speed decreased and pivoting motion occurred more frequently when the number of working legs was reduced by the addition of free sialylated compounds.
| S-EPMC3554017 | biostudies-literature