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Journal of Bacteriology, September 2006, p. 6469-6475, Vol. 188, No. 18
0021-9193/06/$08.00+0     doi:10.1128/JB.00754-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Gliding Motility of Mycoplasma mobile Can Occur by Repeated Binding to N-Acetylneuraminyllactose (Sialyllactose) Fixed on Solid Surfaces

Ryoichiro Nagai1 and Makoto Miyata1,2*

Department of Biology, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan,1 PRESTO, JST, Sumiyoshi-ku, Osaka 558-8585, Japan2

Received 26 May 2006/ Accepted 6 July 2006


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ABSTRACT
 
Mycoplasma mobile relies on an unknown mechanism to glide across solid surfaces including glass, animal cells, and plastics. To identify the direct binding target, we examined the factors that affect the binding of Mycoplasma pneumoniae to solid surfaces and concluded that N-acetylneuraminyllactose (sialyllactose) attached to a protein can mediate glass binding on the basis of the following four lines of evidence: (i) glass binding was inhibited by N-acetylneuraminidase, (ii) glass binding was inhibited by N-acetylneuraminyllactose in a structure-dependent manner, (iii) binding occurred on glass pretreated with bovine serum albumin attached to N-acetylneuraminyllactose, and (iv) gliding speed depended on the density of N-acetylneuraminyllactose on glass.


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INTRODUCTION
 
Mycoplasmas are commensal and occasionally parasitic bacteria with small genomes that lack a peptidoglycan layer (20). Several mycoplasma species form membrane protrusions, such as the head-like structure in Mycoplasma mobile and the attachment organelle in Mycoplasma pneumoniae (11, 15, 24, 25, 27). On solid surfaces, these species exhibit gliding motility in the direction of the protrusion; this motility is believed to be involved in the pathogenicity of mycoplasmas (4, 10, 15). Interestingly, mycoplasmas have no surface flagella or pili and their genomes contain no genes related to known bacterial motility. In addition, no homologs of motor proteins that are common in eukaryotic motility have been found (9, 15).

M. mobile, isolated from the gills of a freshwater fish in the early 1980s, is a fast-gliding mycoplasma (7, 17, 18, 22). It glides smoothly and continuously on glass at an average speed of 2.0 to 4.5 µm/s or three to seven times the length of the cell per s, exerting a force of up to 27 pN. Recently, we identified huge proteins involved in this gliding mechanism (13, 26, 29, 31), visualized the putative machinery and the binding protein (1, 16), and identified the direct energy source used (8, 30). On the basis of these results, we proposed a working model in which cells are propelled by "legs" composed of Gli349 repeatedly binding to and releasing the glass (5, 15, 26, 29) and driven by the force exerted by or through the Gli521 molecule, based on the energy of ATP hydrolysis. This putative mechanism may apply to M. pneumoniae as well, although the protein responsible for glass binding, P1 adhesin, does not share a primary structure with Gli349 of M. mobile. It is because the monoclonal antibody against P1 affects the gliding of M. pneumoniae in a manner very similar to that by which the antibody against Gli349 affects M. mobile gliding (23).

The gliding motility of mycoplasmas can be easily observed on glass surfaces. However, in nature, mycoplasmas live in animal tissues, suggesting that their intrinsic binding target is a surface structure of animal cells or an extracellular matrix. In a previous study, Jaffe et al. found that coating glass with serum is sufficient to allow M. mobile to bind to and glide on glass and that this coating is sensitive to protease treatment (8). These observations suggest that protein components in serum share a structure with those on animal tissues and mediate the glass binding essential for gliding. Identification of the binding targets derived from serum may not be easy, but we should identify the structure which can mediate glass binding to elucidate the gliding mechanism. The net binding of M. pneumoniae monitored by radiolabeling has been reported to depend on both sulfated glycolipids and N-acetylneuraminic acid (the representative of sialic acids), but their roles in the binding of individual cells and in gliding were not examined (12, 21).

In this study, we examined the materials involved in the net binding of M. pneumoniae and concluded that N-acetylneuraminyllactose can mediate the glass binding required for M. mobile gliding.


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MATERIALS AND METHODS
 
Materials. The following three compounds were purchased from Dextra Laboratories Ltd. (Reading, United Kingdom): 3'-N-acetylneuraminyllactose (3'-sialyllactose; Neu5Ac{alpha}2-3Galß1-4Glc), 6'-N-acetylneuraminyllactose (6'-sialyllactose; Neu5Ac{alpha}2-6Galß1-4Glc), and 3'-SLN-BSA (3'-N-acetyllactosamine-BSA, Neu5Ac{alpha}2-3Galß1-4GlcNAc-three-atom spacer-NH-lysine-bovine serum albumin). Dextran sulfate (Mrs, 500,000 and 5,000) and N-acetylneuraminidase (Clostridium perfringens, type V) were purchased from Sigma.

Cultivation. M. mobile strain 163K (ATCC 43663) and its mutants were grown at 25°C in Aluotto medium, consisting of 2.1% heart infusion broth, 0.56% yeast extract, 10% horse serum, 0.025% thallium acetate, and 0.005% ampicillin (2, 19).

Measurement of cell binding and gliding speed. M. mobile cells at an optical density at 600 nm of 0.03 to 0.07 were collected by centrifugation at 12,000 x g for 4 min and suspended in the medium or a buffer so as to reach an optical density of 1.0, corresponding to 7 x 109 CFU/ml. To examine the effects of serum, cells were washed once with phosphate-buffered saline (PBS)-G buffer (137 mM NaCl, 8.1 mM sodium phosphate [pH 7.4], 2.68 mM KCl, 1.47 mM potassium phosphate, 10 mM glucose) and suspended in the solutions tested. The cell suspension was inserted into a tunnel chamber (2-mm interior width, 18-mm length, 60-µm wall thickness) constructed with a coverslip and a glass slide and assembled with double-sided tape (13, 29). Mycoplasmas bound to the coverslip were observed with a microscope with a 100x phase-contrast objective lens (BX50; Olympus, Tokyo, Japan) and recorded with a DVD video recorder (DMR-E30; Panasonic, Kadoma, Japan) through a charge-coupled device camera (BP510; Panasonic). Two-second movies were converted to stacks of three frames per second with Video Editor version 6.5 (Ulead Systems Inc., Taipei, Taiwan). To examine the fluctuation of gliding speed, movies were converted to stacks of 10 frames per second. Gliding speeds were calculated as described previously and averaged for 30 cells and are presented along with standard deviations if not otherwise mentioned (17). The cells bound to a video field of 320 µm2 were counted, and the number is presented as the ratio of cells bound to the whole coverslip surface relative to the initial total number of cells inserted into the tunnel chamber (26, 29). Bound-cell numbers are presented as averages of three measurements along with the standard deviations.

Coating of a glass surface with 3'-SLN-BSA (3'-N-acetyllactosamine-BSA) or BSA. Coverslips were cleaned with ethanolic KOH as described previously (3, 17), subjected to coating with various concentrations of protein solutions at room temperature for 1 h, and dried in a laminar-flow station.


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RESULTS
 
Measurement of glass binding by tunnel chamber assay. A tunnel chamber composed of a coverslip and a glass slide and assembled with double-sided tape was used to examine the binding of M. mobile cells to glass surfaces (13, 29). M. mobile cells were collected by centrifugation and suspended in PBS-G buffer. The cell suspension was inserted into a tunnel chamber, and the cells bound to the glass coverslip were counted at various times (Fig. 1). The mycoplasma cells started to bind to the glass surface from time zero, and the bound-cell ratio increased to a plateau, where association of cells with glass and dissociation of cells from glass were in equilibrium, at around 300 s. Therefore, in the following experiments, we measured the bound-cell ratio and gliding speed at 360 s after insertion.


Figure 1
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  		FIG. 1. Time course of cell binding in a tunnel chamber assay. Mycoplasma cells suspended in PBS-G with 10% horse serum were inserted into a tunnel chamber. The cells bound to the coverslip were counted at each time point, and the results are presented as a ratio relative to the total number of cells in the tunnel chamber.

Serum is required for glass binding. Coating of a glass surface with protein components of serum has been reported as necessary for glass binding for M. mobile gliding (8). To obtain more information about this observation, we examined different concentrations of serum and different strains (Fig. 2). The m13 strain has a missense mutation in the gli349 gene, whose product is responsible for glass binding for gliding, and this mutant exhibits a nonbinding phenotype in growth medium (19, 29). The bound-cell numbers of the wild-type strain were determined separately for gliding and nongliding cells. The ratio of bound and gliding cells increased with the serum concentration and reached a plateau at 10% serum. This observation suggests that serum is essential to glass binding for gliding. On the other hand, binding without gliding showed 1.7% and 1.5% ratios at 0% serum for the wild-type and m13 strains, respectively. Considering that the binding site of Gli349 is lacking in the m13 strain (1, 29), the binding observed in m13 should not be related to gliding. This "nonspecific binding" was reduced by the addition of serum to 0.22% at 10% serum, showing that serum components can reduce nonspecific binding. The binding ratio of the wild-type strain without gliding, 1.6% at 0% serum, was reduced to 0.06% at 10% serum, suggesting that nonspecific binding by the wild-type strain occurs as well and is reduced by the components in serum.


Figure 2
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  		FIG. 2. Effects of serum on glass binding of cells of the wild type (WT) and the m13 mutant truncated for Gli349. Cells suspended in PBS-G containing various concentrations of serum were poured into a tunnel chamber, and the cells bound to the glass were counted after incubation for 600 s. The bound-cell ratio is presented relative to the total number of cells in the tunnel chamber.

Inhibition of glass binding by N-acetylneuraminidase. To examine the possibility that M. mobile binds to glass via glycoproteins containing N-acetylneuraminic acid residues, we examined the effects of N-acetylneuraminidase (sialidase) on gliding mycoplasmas. This enzyme hydrolyzes linkages of terminal sialic acid residues, including N-acetylneuraminyl-{alpha}(2-3)-lactose, N-glycolylneuraminyl-{alpha}(2-3)-lactose, N-acetylneuraminyl-{alpha}(2-6)-lactose, and N-acetyl-9-O-acetylneuraminyl-{alpha}(2-3)-lactose (28). We inserted the cell suspension into the tunnel chamber and then replaced the medium with PBS-G buffer after 360 s of incubation. The PBS-G buffer was then replaced with a buffer containing N-acetylneuraminidase. The number of cells bound to the surface decreased to 29.1% of the initial value at 360 s after enzyme addition and to 3.4% at 660 s, although 68.4% of the cells remained at 660 s when the enzyme was not added (Fig. 3A). The decrease without the enzyme was likely caused by the removal of cells suspended in the tunnel chamber because the equilibrium between association and dissociation was lost when the buffer was replaced. The gliding speed decreased and reached around 65% of the initial value at about 200 s, although the ratio fell to only 81% when the enzyme was not applied (Fig. 3B). These results suggest that terminal sialic acids are essential for binding during gliding and that the decrease in their density on the glass surface affects their gliding speed.


Figure 3
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  		FIG. 3. Effects of N-acetylneuraminidase on glass binding and gliding speed. N-Acetylneuraminidase was added to cells gliding in a tunnel chamber, to 60 µg/ml. (A) Inhibition of glass binding. Numbers of bound cells are presented as ratios relative to the number at time zero. (B) Inhibition of gliding speed. Gliding speed is presented as a ratio relative to the initial gliding speed. Closed and open circles present results obtained with and without N-acetylneuraminidase, respectively.

Inhibition of binding and gliding by N-acetylneuraminyllactose. The glass binding of M. pneumoniae is known to be inhibited by 3'-N-acetylneuraminyllactose (3'-sialyllactose; Neu5Ac{alpha}2-3Galß1-4Glc) and 6'-N-acetylneuraminyllactose (6'-sialyllactose; Neu5Ac{alpha}2-6Galß1-4Glc), namely, N-acetylneuraminic acid {alpha}2-3 linked and {alpha}2-6 linked to lactose, respectively (21). Therefore, we examined the effects of these compounds at 0.05 to 2 mM on glass binding and gliding speed in a tunnel chamber (Fig. 4). We replaced the PBS-G buffer with PBS-G containing 0.05 to 2 mM N-acetylneuraminyllactose. Both 3'-N-acetylneuraminyllactose and 6'-N-acetylneuraminyllactose inhibited glass binding during gliding in a concentration-dependent manner. The number of bound cells decreased after the addition of N-acetylneuraminyllactose. 3'-N-Acetylneuraminyllactose reduced the number of bound cells by 50% in 0.8 s at 2 mM, in 2.8 s at 0.5 mM, and in 22 s at 0.2 mM. 6'-N-Acetylneuraminyllactose also inhibited their binding but required a concentration about twofold higher than that of 3'-N-acetylneuraminyllactose for the same degree of inhibition, suggesting that {alpha}2-3 has a higher affinity for M. mobile cells than {alpha}2-6 does. We examined 2 mM lactose, maltose, galactose, N-acetylneuraminic acid, and D-glycoylneuraminic acid and found no significant effects. These results suggest that this inhibition is specific for N-acetylneuraminyllactose and that M. mobile binds to N-acetylneuraminyllactose rather than to N-acetylneuraminic acid. M. pneumoniae also binds to sulfated glycolipids, and the binding is inhibited by dextran sulfate (12). We examined the effects of dextran sulfate on the binding and gliding of M. mobile and found that it has no effect, even at 2 mM.


Figure 4
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  		FIG. 4. Inhibitory effects of N-acetylneuraminyllactose on glass binding and gliding speed. (A) Number of cells bound to a glass surface after addition of 3'-N-acetylneuraminyllactose. The number of cells on the glass surface at each time point is presented as a ratio relative to that at time zero. (B) Gliding speed after addition of various concentrations of 3'-N-acetylneuraminyllactose. (C) Glass binding after addition of various concentrations of 6'-N-acetylneuraminyllactose. (D) Gliding speed after addition of various concentrations of 6'-N-acetylneuraminyllactose. The millimolar concentrations of N-acetylneuraminyllactose applied are indicated in panel D. The same set of concentrations was used for both compounds.

3'-SLN-BSA can mediate glass binding for gliding. To demonstrate that N-acetylneuraminyllactose can mediate the glass binding of M. mobile for gliding, we examined the activity for glass binding of 3'-SLN-BSA, which is BSA attached to 3'-N-acetylneuraminyllactose via a linker. Mycoplasma cells of the wild-type strain or the m13 mutant were inserted into a tunnel chamber assembled with a coverslip coated with 3'-SLN-BSA and observed after 360 s (Fig. 5). The number of bound and gliding cells of the wild-type strain increased in accordance with the concentration of 3'-SLN-BSA and reached a maximum at 0.1 µg/ml (Fig. 5A). These results show that N-acetylneuraminyllactose attached to BSA mediates glass binding for gliding and that this protein reduces glass binding unrelated to gliding. Significant numbers of nongliding cells bound to glass when the concentration of 3'-SLN-BSA or BSA was low. Glass binding by nongliding cells of strain m13 was also observed (Fig. 5B). The numbers of nonspecifically bound cells were decreased by the addition of 3'-SLN-BSA or BSA, suggesting that these proteins can reduce nonspecific binding as observed for serum (Fig. 2).


Figure 5
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  		FIG. 5. Binding of cells to glass pretreated with 3'-SLN-BSA or BSA. Coverslips were pretreated with various concentrations of 3'-SLN-BSA or BSA. Numbers of cells bound to the glass are presented as ratios relative to the total number of cells in the tunnel chamber. (A) Wild-type (WT) cells. (B) Strain m13 mutant cells.

Gliding speed and stability depend on the density of 3'-SLN-BSA on glass. If mycoplasma cells actually walk on 3'-SLN-BSA, their gliding speed should depend on the density of 3'-SLN-BSA. We then measured their speed at various concentrations of 3'-SLN-BSA to examine the effect of the density of the binding target on gliding speed (Fig. 6A). Gliding speed depended on the concentration of 3'-SLN-BSA, with a peak at 30 ng/ml. At lower concentrations of 3'-SLN-BSA, cells were sometimes stuck on the glass surface during gliding. We then examined the fluctuation of gliding speed caused by treatment with 3'-SLN-BSA at different densities by measuring gliding speed every 0.1 s on surfaces pretreated with 3 ng/ml and 10 µg/ml 3'-SLN-BSA (Fig. 6B). Gliding speed was more stable with 10 µg/ml 3'-SLN-BSA. Mycoplasma cells were often stuck on the surface with 3 ng/ml 3'-SLN-BSA. We compared the distributions of gliding speeds at various densities (Fig. 6C). A comparison by F test of standard deviations of gliding speed showed significant differences (P < 0.05) among concentrations of 3 to 10 ng/ml, 0.03 to 1 µg/ml, and 10 µg/ml, suggesting that gliding speed depends on the opportunity of the leg protein Gli349 to bind to N-acetylneuraminyllactose at a new position.


Figure 6
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  		FIG. 6. Fluctuation of gliding speed at various densities of 3'-SLN-BSA. (A) Gliding speeds measured every 2 s on glass pretreated with various concentrations of 3'-SLN-BSA. (B) Gliding speeds measured every 0.1 s starting from 0.05 s earlier than the time indicated are presented by closed and open symbols for surfaces pretreated with 10 µg/ml 3'-SLN-BSA and 3 ng/ml 3'-SLN-BSA, respectively. Three traces from different cells are presented by different symbols for each condition. (C) Distribution of gliding speeds on glass pretreated with various concentrations of 3'-SLN-BSA. Averages and standard deviations are indicated by closed and open triangles, respectively.


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DISCUSSION
 
N-Acetylneuraminyllactose mediates glass binding. We previously showed that a leg protein, Gli349, localizing at the cell neck is responsible for glass binding during gliding (29). The next focus for the investigation of glass binding should be the direct binding target of this protein. Jaffe et al. showed that protein components in horse serum mediate the binding of M. mobile for gliding (8). In the present study, we showed that N-acetylneuraminyllactose attached to a protein can mediate glass binding, on the basis of the following four lines of evidence: (i) glass binding was inhibited by N-acetylneuraminidase, (ii) glass binding was inhibited by N-acetylneuraminyllactose in a structure-dependent manner, (iii) binding occurred on glass pretreated with BSA attached to N-acetylneuraminyllactose, and (iv) gliding speed depended on the density of N-acetylneuraminyllactose on glass.

We concluded that M. mobile binds to targets similar to those used for binding by M. pneumoniae. Previous studies of the binding target of M. pneumoniae did not examine the binding and gliding of individual cells (12, 21), but it is possible that M. pneumoniae also binds to the targets used for gliding. The amino acid sequence of the binding protein of M. pneumoniae, P1 adhesin, does not show any similarities to M. mobile Gli349 (1, 14, 29). However, the three-dimensional structure around the binding sites may be very similar. The failure of sulfated glycolipids to affect M. mobile binding may reflect the differences in the structures of the binding proteins. Alternatively, the binding of M. pneumoniae to dextran sulfate may be caused by another protein (21).

Gliding motility was observed on various surfaces. M. mobile cells showed binding to and gliding on solid surfaces with speeds similar to those on glass, including animal cells, plastics, cellulose nitrate film, and mica (data not shown). These observations can be explained by the assumption that the components of serum contained in the medium were adsorbed to the solid surfaces and mediated glass binding. When a positive charge was introduced onto the glass surface by modification with positively charged compounds, glass binding was lost (data not shown). This result can be explained by assuming that adsorption of protein components attached to N-acetylneuraminyllactose does not occur on positively charged surfaces.

Nonspecific binding to the surface. We found that M. mobile cells show glass binding unrelated to gliding when the buffer does not contain serum or proteins (Fig. 2 and 5). This nonspecific binding was reduced by the addition of serum, BSA, or 3'-SLN-BSA. Presumably, structures other than Gli349 on the cell surface adsorb to the glass surface, as is observed for protein adsorption to clean glass. The mechanism may be the same as that by which glycoproteins containing N-acetylneuraminyllactose in serum adsorb to solid surfaces.

Gliding speed. Both average gliding speed and gliding stability depended on the density of 3'-SLN-BSA on glass, showing that this complex provides the actual binding target for gliding (Fig. 6). The average gliding speed was highest for the 30-ng/ml treatment of glass and lower after both lower- and higher-concentration treatments (Fig. 6A). We have proposed a working model for the gliding mechanism as a repeated cycle that propels the cell, including the following steps: (i) initial binding, (ii) tight binding, (iii) stroke, (iv) movement, (v) release, and (vi) return (15, 26, 29). The results reported here can be explained by considering that step i is dependent on the density of the binding target on glass and can be rate limiting. It requires more time at lower densities of 3'-SLN-BSA, while binding of the leg occurs at smaller distance intervals at higher densities. This assumption is supported by the observation that gliding speed fluctuates more at lower densities.

Binding domain of Gli349. Recently, isolation of Gli349, electron microscopy, and amino acid sequence analyses have shown the outline of this protein (1, 14). The protein molecule typically has a musical-note-like morphology, is about 100 nm long, and features a terminal globule called a "foot" connected with three rods 45, 20, and 20 nm long. Its amino acid sequence features 18 weak repeats of about 100 amino acids and can be assigned to a molecular image by electron microscopy from the rod end to the foot end. As a transmembrane segment is predicted at the N terminus, the rod end is likely embedded in the membrane and the C terminus at the distal end. Therefore, we examined the similarity of the amino acid sequence of Gli349, especially focusing on the C-terminal region, with sialic acid binding proteins. However, no features related to sialic acid binding proteins were identified. This attempt failed for the whole sequence of the P1 adhesin of M. pneumoniae. These facts suggest that the binding proteins of these two species belong to novel types of sialic acid binding protein.

Prospects. In this study, we clarified the mechanism by which M. mobile glides on a wide variety of solid surfaces, including many materials quite different from the natural substrates. The identification of the binding target for gliding not only gives us a clue for elucidating the gliding mechanism but also offers flexibility in designing experiments. There has also been an attempt to make a "micro" hybrid motor of material and bacterial cells by controlling the gliding direction of M. mobile with a micropatterning technology whereby the gliding direction is controlled by "wall" and "cliff" mechanisms (6, 7). Now, a "route" mechanism is available as another method to guide gliding mycoplasmas (6a).


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ACKNOWLEDGMENTS
 
For helpful discussions, we are grateful to Yuichi Hiratsuka at Tokyo University and Atsuko Uenoyama at Osaka City University.

This work was supported by a Grant-in-Aid for Scientific Research on the Priority Areas "Applied Genomics" and "Structures of Biological Macromolecular Assemblies" from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to M.M.) and by a grant from the Institution for Fermentation Osaka (to M.M.).


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FOOTNOTES
 
* Corresponding author. Mailing address: PRESTO, JST, Sumiyoshi-ku, Osaka 558-8585, Japan. Phone: 81 (6) 6605 3157. Fax: 81 (6) 6605 3158. E-mail: miyata{at}sci.osaka-cu.ac.jp. Back


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Journal of Bacteriology, September 2006, p. 6469-6475, Vol. 188, No. 18
0021-9193/06/$08.00+0     doi:10.1128/JB.00754-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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