Identification of novel protein domain for sialyloligosaccharide binding essential to Mycoplasma mobile gliding.
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ABSTRACT:
SUBMITTER: Hamaguchi T
PROVIDER: S-EPMC6376172 | biostudies-literature | 2019 Feb
REPOSITORIES: biostudies-literature
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Project description:Several mycoplasma species are known to glide in the direction of the membrane protrusion (head-like structure), but the mechanism underlying this movement is entirely unknown. To identify proteins involved in the gliding mechanism, protein fractions of Mycoplasma mobile were analyzed for 10 gliding mutants isolated previously. One large protein (Gli349) was observed to be missing in a mutant m13 deficient in hemadsorption and glass binding. The predicted amino acid sequence indicated a 348,758-Da protein that was truncated at amino acid residue 1257 in the mutant. Immunofluorescence microscopy with a monoclonal antibody showed that Gli349 is localized at the head-like protrusion's base, which we designated the cell neck, and immunoelectron microscopy established that the Gli349 molecules are distributed all around this neck. The number of Gli349 molecules on a cell was estimated by immunoblot analysis to be 450 +/- 200. The antibody inhibited both the hemadsorption and glass binding of M. mobile. When the antibody was used to treat gliding mycoplasmas, the gliding speed and the extent of glass binding were inhibited to similar extents depending on the concentration of the antibody. This suggested that the Gli349 molecule is involved not only in glass binding for gliding but also in movement. To explain the present results, a model for the mechanical cycle of gliding is discussed.
| S-EPMC344404 | 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:A protein with a molecular mass of 42 kDa (P42) from Mycoplasma mobile, one of several mycoplasmas that exhibit gliding motility, was shown to be a novel NTPase (nucleoside triphosphatase). Although the P42 protein lacks a common ATP-binding sequence motif (Walker A), the recombinant proteins expressed in Escherichia coli certainly hydrolysed some nucleoside triphosphates, including ATP. The results of photoaffinity labelling by an ATP analogue supported that the P42 protein contains a specific binding site for ATP (or another nucleoside triphosphate). In the M. mobile genome, the P42 gene is located downstream of gli123, gli349 and gli521 genes, and they have been reported to be polycis-tronically transcribed. As the huge proteins encoded by gli123, gli349 and gli521 play a role in gliding motility of M. mobile, P42 might also have some kind of function in the gliding motility. The gliding motility of M. mobile is driven directly by ATP hydrolysis, but the key ATPase has not been identified. Our results showed that, among these four proteins, only P42 exhibited ATPase activity. Biochemical characteristics--optimal conditions for activity, substrate specificities, and inhibiting effects by ATP analogues--of the recombinant P42 proteins were very similar to those of a putative ATPase speculated from a previous analysis with a gliding 'ghost' whose cell membrane was permeabilized by Triton X-100. These results support the hypothesis that the P42 protein is the key ATPase in the gliding motility of M. mobile.
| S-EPMC1828890 | biostudies-other
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:Among the bacteria that glide on substrate surfaces, Mycoplasma mobile is one of the fastest, exhibiting smooth movement with a speed of 2.0-4.5 μm⋅s(-1) with a cycle of attachment to and detachment from sialylated oligosaccharides. To study the gliding mechanism at the molecular level, we applied an assay with a fluorescently labeled and membrane-permeabilized ghost model, and investigated the motility by high precision colocalization microscopy. Under conditions designed to reduce the number of motor interactions on a randomly oriented substrate, ghosts took unitary 70-nm steps in the direction of gliding. Although it remains possible that the stepping behavior is produced by multiple interactions, our data suggest that these steps are produced by a unitary gliding machine that need not move between sites arranged on a cytoskeletal lattice.
| S-EPMC4060671 | biostudies-other
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
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:UnlabelledMycoplasma mobile, a fish-pathogenic bacterium, features a protrusion that enables it to glide smoothly on solid surfaces at a velocity of up to 4.5 µm s(-1) in the direction of the protrusion. M. mobile glides by a repeated catch-pull-release of sialylated oligosaccharides fixed on a solid surface by hundreds of 50-nm flexible "legs" sticking out from the protrusion. This gliding mechanism may be explained by a possible directed binding of each leg with sialylated oligosaccharides, by which the leg can be detached more easily forward than backward. In the present study, we used a polystyrene bead held by optical tweezers to detach a starved cell at rest from a glass surface coated with sialylated oligosaccharides and concluded that the detachment force forward is 1.6- to 1.8-fold less than that backward, which may be linked to a catch bond-like behavior of the cell. These results suggest that this directed binding has a critical role in the gliding mechanism.ImportanceMycoplasma species are the smallest bacteria and are parasitic and occasionally commensal, as represented by Mycoplasma pneumoniae, which causes so-called "walking pneumonia" in humans. Dozens of species glide on host tissues, always in the direction of the characteristic cellular protrusion, by novel mechanisms. The fastest species, Mycoplasma mobile, catches, pulls, and releases sialylated oligosaccharides (SOs), which are common targets among influenza viruses, by means of a specific receptor based on the energy of ATP hydrolysis. Here, force measurements made with optical tweezers revealed that the force required to detach a cell from SOs is smaller forward than backward along the gliding direction. The directed binding should be a clue to elucidate this novel motility mechanism.
| S-EPMC4937208 | biostudies-literature
Project description:The cell wall-less prokaryote Mycoplasma pneumoniae causes bronchitis and atypical pneumonia in humans. Mycoplasma attachment and gliding motility are required for colonization of the respiratory epithelium and are mediated largely by a differentiated terminal organelle. P30 is a membrane protein at the distal end of the terminal organelle and is required for cytadherence and gliding motility, but little is known about the functional role of its specific domains. In the current study, domain deletion and substitution derivatives of P30 were engineered and introduced into a P30 null mutant by transposon delivery to assess their ability to rescue P30 function. Domain deletions involving the extracellular region of P30 severely impacted protein stability and adherence and gliding function, as well as the capacity to stabilize terminal organelle protein P65. Amino acid substitutions in the transmembrane domain revealed specific residues uniquely required for P30 stability and function, perhaps to establish correct topography in the membrane for effective alignment with binding partners. Deletions within the predicted cytoplasmic domain did not affect P30 localization or its capacity to stabilize P65 but markedly impaired gliding motility and cytadherence. The larger of two cytoplasmic domain deletions also appeared to remove the P30 signal peptide processing site, suggesting a larger leader peptide than expected. We propose that the P30 cytoplasmic domain may be required to link P30 to the terminal organelle core, to enable the P30 extracellular domain to achieve a functional conformation, or perhaps both.
| S-EPMC3067664 | biostudies-literature