Force and step size of gliding motility in human pathogenic bacterium Mycoplasma pneumoniae

Mycoplasma pneumoniae, a human pathogenic bacterium, binds to sialylated oligosaccharides and glides on host cell surfaces via a unique mechanism. Gliding motility is essential for initiating the infectious process. In the present study, we measured the stall force of an M. pneumoniae cell carrying a bead that was manipulated using optical tweezers on two strains. The stall forces of M129 and FH strains were averaged to be 23.7 and 19.7 pN, respectively, much weaker than those of other bacterial surface motilities. The binding activity and gliding speed on sialylated oligosaccharides of the M129 strain were eight and two times higher than those of the FH strain, respectively, showing that binding activity is not linked to gliding force. Gliding speed decreased when cell binding was reduced by addition of free sialylated oligosaccharides, indicating the existence of a drag force during gliding. We detected stepwise movements under 0.2–0.3 mM free sialylated oligosaccharides. A step size of 14–19 nm showed that 25–35 propulsion steps per second are required to achieve the usual gliding speed. The step size was reduced to less than half with the load applied using optical tweezers, showing that a 2.5 pN force from a cell is exerted on a leg. The work performed in this step was 16–30% of the free energy of the hydrolysis of ATP molecules, suggesting that this step is linked to the elementary process of M. pneumoniae gliding. Significance Statement Human mycoplasma pneumonia is caused by the bacterium Mycoplasma pneumoniae. This tiny bacterium, shaped like a missile, binds to human epithelial surfaces and spreads using a unique gliding mechanism to establish infection. Here, we analyzed the movements and force of this motility using a special setup: optical tweezers. We then obtained detailed mechanical data to understand this mechanism. Furthermore, we succeeded in detecting small steps of nanometers in its gliding, which is likely linked to the elementary process of the core reaction: chemical to mechanical energy conversion. These data provide critical information to both control this human pathogen and explore new ideas for artificial molecular machines.

cell surfaces related to cell-cell recognition, and the binding targets of many pathogens and toxins 66 (7-10). The cells show unidirectional gliding motility at a speed of up to 1 μm/s on SOs-coated 67 glass surfaces (Fig. 1A) (5, 11), which is known to be essential for their infection (12). The gliding 68 machinery, called the "attachment organelle," is localized at a cell pole (13). The attachment 69 organelle is divided into two parts: internal and surface structures. The internal structure is 70 composed of an internal core complex and a surrounding translucent area. The internal core 71 comprises three parts: a terminal button, paired plates, and a bowl complex from the front side of 72 the cell (Fig. 1B) (5, 14, 15). The major surface structure, called "P1 adhesin complex" or 73 "genitalium and pneumoniae cytoadhesin (GPCA)", is composed of P1 adhesin and P40/P90 74 proteins and aligned around the internal structure, which plays a dual role as the adhesin to bind 75 to SOs and as the leg for gliding (Fig. 1B) (16-20). The model for gliding called the "Inchworm 76 model" or "Double-spring hybrid ratchet model" is proposed, in which cells repeat the extensions 77 and contractions of the attachment organelle based on the energy from ATP hydrolysis to enable 78 smooth gliding (15, 21-23). Generally, the mechanical characteristics and detailed analysis of 79 movements are essential for creating and completing a detailed model for the motility 80 mechanism. However, to date, no information is available about the force for gliding.

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In the present study, we measured the stall forces of the two strains and discuss the 82 relationship between binding and force. Furthermore, we succeeded in detecting and measuring 83 stepwise movements that are likely linked to the elementary process of the gliding reaction.

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Stall force measurement using optical tweezers

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Optical tweezers are commonly used to measure the stall force generated by pili in bacterial 88 motility or motor proteins in eukaryotic motility (24-27). The stall force is defined as the force 89 needed to stop movements and is equal to the maximal propulsion force for locomotion.

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Previously, we measured the stall force for the gliding motility of M. mobile, based on a 91 mechanism unrelated to M. pneumoniae gliding, using optical tweezers (28, 29). In the present 92 study, we applied this method to M. pneumoniae gliding. M. pneumoniae cells were biotinylated 93 and inserted into a tunnel chamber, which was assembled using two glass plates and double-94 sided tape (30). An avidin-conjugated polystyrene bead was trapped by a highly focused laser 95 beam and attached to a gliding cell at the back end of the cell body by exploiting the avidin-biotin 96 interaction. The cells pulled the bead from the trap center with gliding and then stalled ( Fig. 2A   97 and B; SI Appendix, Movie S1). The force was calculated by measuring the distance between the 98 centers of the bead and the laser trap, which was multiplied by the trap stiffness; the force acting 99 on the bead increased linearly with the displacement from the trap center (24). Starting from 0 s, 100 the pulling force increased and reached a plateau in 120 s (Fig. 2C). The maximal value of the 101 force averaged over one second was determined as the stall force. The stall force of M129 strain 102 cells was 23.7 ± 6.3 pN (Fig. 2E). We also measured the stall force of the FH strain, another 103 major strain of M. pneumoniae. The cells of the FH strain pulled the bead in a manner similar to 104 that of M129 cells (SI Appendix, Movie S2). The stall force of the FH strain was 19.7 ± 5.3 pN, 105 significantly weaker than that of the M129 strain ( Fig. 2D and E). To characterize the differences in gliding motility, we measured the binding activity and gliding 109 speed of the M129 and FH strains. M. pneumoniae cells were suspended in HEPES buffer 110 containing 20 mM glucose to obtain an optical density at 595 nm of 0.07, then inserted into tunnel 111 chambers. After incubation for 5-30 min, the tunnel chambers were washed and observed by 112 phase-contrast microscopy. The number of bound cells in the M129 strain increased with time 113 from 90 ± 16 to 308 ± 20 cells in 100 ×100 μm 2 area from 5 to 30 min ( Fig. 3A and B). These 114 values are consistent with the results of previous report (9). In contrast, the number of bound cells 115 in the FH strain increased from 12 ± 2 to 36 ± 8 cells from 5 to 30 min ( Fig. 3A and B). The 116 number of bound cells in the FH strain was 7.2-8.7-fold smaller than that of the M129 strain at all 117 time points (Fig. 3B). When 10× concentrated cell suspension was examined, the bound cell 118 numbers of the FH strain increased from 117 ± 10 to 386 ± 24 cells from 5 to 30 min, 1.2-1.3 119 times that of M129 at all time points (Fig. 3A and B). These results indicate that the FH strain has 120 approximately 8-fold lower binding activity to SOs-coated glass surfaces than the M129 strain.

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Considering that the stall force of FH was only 1.2-fold smaller than that of the M129 strain, the 122 force is unlikely to be linked to binding activity.  The FH strain used in this study has not been genomically analyzed. Therefore, we sequenced 133 the genomes of both strains using MiSeq (SI Appendix, Dataset S1) and analyzed the sequences

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The addition of free SL slowed down and then stopped gliding or released the gliding cells from 156 the glass surfaces, but did not release non-gliding cells, indicating that release requires glass 157 binding by GPCA with displacements (Fig. 4A). The number of gliding cells and the gliding speed 158 relative to the initial speed were decreased by 0.1-0.5 mM with SL treatments from 55 ± 11% to 159 26 ± 16% and from 0.33 ± 0.06 to 0.19 ± 0.07 μm/s, respectively ( Fig. 4B and C). We then        217 pneumoniae (23). The decrease in speed was probably caused by the drag force generated from 218 the substrate surface, because the friction force exerted from water is estimated to be more than 219 5000 times smaller than the stall force of 24 pN (Fig. 2E) (36, 37). As the cause of the drag force,

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The attachment organelle responsible for gliding can be divided into a surface structure 274 including GPCA and an internal rod structure (15, 22, 50-52). Briefly, the force for gliding 275 is likely generated at the bowl because a few proteins essential for gliding and not binding

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(GPCA), due to the lack of information about step, force, and foot structure. In this study, we 305 succeeded in adding new information and completing an updated model.