s2fieldsofstudy: Biology Engineering

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Altered motility of Caulobacter Crescentus in viscous and viscoelastic media

Background Motility of flagellated bacteria depends crucially on their organelles such as flagella and pili, as well as physical properties of the external medium, such as viscosity and matrix elasticity. We studied the motility of wild-type and two mutant strains of Caulobacter crescentus swarmer cells in two different types of media: a viscous and hyperosmotic glycerol-growth medium mixture and a viscoelastic growth medium, containing polyethylene glycol or polyethylene oxide of different defined sizes. Results For all three strains in the medium containing glycerol, we found linear drops in percentage of motile cells and decreases in speed of those that remained motile to be inversely proportional to viscosity. The majority of immobilized cells lost viability, evidenced by their membrane leakage. In the viscoelastic media, we found less loss of motility and attenuated decrease of swimming speed at shear viscosity values comparable to the viscous medium. In both types of media, we found more severe loss in percentage of motile cells of wild-type than the mutants without pili, indicating that the interference of pili with flagellated motility is aggravated by increased viscosity. However, we found no difference in swimming speed among all three strains under all test conditions for the cells that remained motile. Finally, the viscoelastic medium caused no significant change in intervals between flagellar motor switches unless the motor stalled. Conclusion Hyperosmotic effect causes loss of motility and cell death. Addition of polymers into the cell medium also causes loss of motility due to increased shear viscosity, but the majority of immobilized bacteria remain viable. Both viscous and viscoelastic media alter the motility of flagellated bacteria without affecting the internal regulation of their motor switching behavior. Electronic supplementary material The online version of this article (doi:10.1186/s12866-014-0322-3) contains supplementary material, which is available to authorized users.

Background
Many gram-negative bacteria depend on appendages such as flagella, pili, and stalks for successful colonization of their environment. It is well known that the mechanical environment can trigger changes in gene expression and biochemical signaling, and subsequently, induce a functional response from the cells [1]. Studies also have shown, however, some aspects of the mechanical regulation are independent of feedback from genetic or biochemical signaling pathways. Examples include the motor's sensitivity to load in E. coli [2,3] and the flicking motion of a uni-flagellated bacterium as it changes swimming direction from backward to forward, caused by a buckling instability in the hook [4,5]. These recent findings motivate testing of other scenarios whereby mechanical properties of the medium directly dictate or alter motility-related functions of a motile bacterium. Such testing requires a convenient, mechanical platform upon which more refined studies may be designed, and through this line of study certain properties of mechanical origin may be sorted out from others due to genetic regulation or biochemical signaling.
Numerous chemical agents have been utilized experimentally to alter the mechanical environment of bacteria, especially impacting their motility. Studies performed decades ago explored the effects of elevated viscosity on the swimming speed of several species of bacteria, using extremely high molecular weight polymers such as methylcellulose and polyvinylpyrrolidone (PVP) [6][7][8]. It was then recognized that the "gel-like", or weakly elastic nature of these polymers accounted for enhanced motility of flagellated bacteria [9][10][11][12]. Another study shows that for Vibrio alginolyticus, the swimming speed of a mutant strain (YM4) expressing only a polar flagellum decreases with increased viscosity in PVP containing media, whereas the strains expressing lateral flagella increase swimming speed with viscosity and attain a peak speed at around 5 cP [13].
From the polymer rheology perspective, the balance between the viscous and elastic effects is highly dependent on the frequency of the mechanical perturbance, which is periodically exerted by the rotating flagella and cell body [14,15]. The so-called frequency-dependent viscoelasticity is generally dictated by polymer size, concentration, and network entanglement [16,17]. It is therefore most informative to characterize the mechanical effects of polymers of well-defined structure and size on bacterial motility. Our choice for this study is the inert polyethylene glycol (PEG) or polyethylene oxide (PEO) of linear structure and with a wide range of sizes available. PEG and PEO are nearly identical polymers, but differentially termed depending on whether the molecular weight is below or above 100,000 daltons.
Mechanical inhibition of flagellar motor rotation, due to elevated medium viscosity or proximity to surfaces, is also known to trigger swarmer-cell differentiation, which is a key step in pathogenesis in some bacterial species [18][19][20][21]. Specifically, recent work on Caulobacter crescentus shows that jamming of the flagellum rotation caused by pili-mediated surface contact or aggregation of cells induced by polymeric crowding agents triggers a just-intime secretion of surface adhesins [22]. In light of this finding, we choose Caulobacter crescentus as a model species to explore for effects on motility caused by mechanical inhibitions that are distinct in nature and perhaps sensitive to the type and molecular size of viscous agents used.
Caulobacter crescentus is a gram-negative, αproteobacterium often found in nutrient-poor, freshwater environments [23][24][25]. It has a dimorphic life cycle, spending time both as a non-replicating, motile swarmer cell and as a replicating, sessile, stalked cell. During the swarmer stage of its life, it is uni-flagellated. The rotation of its 5-10 μm long flagellum is entirely responsible for moving its 1-2 μm long crescent-shaped body. The swarmer bacterium eventually matures into a sessile cell, characterized by a long, thin, and rigid cell envelope extension called a stalk [26], which sticks to a nearby surface via an elastic, polysaccharide-based, adhesive holdfast [27][28][29]. This attachment process is facilitated by their type-IV pili, which extend a few μm from the same pole as the flagellum [30,31], and can retract upon sticking to a surface, or binding of a bacteriophage [32].
In this study we report on the motility and swimming behavior of C. crescentus and its two mutant strains lacking pili in two contrasting media: the Newtonian, i.e., purely viscous, glycerol-water mixture and a viscoelastic medium containing the flexible polymer of PEG or PEO. The wild-type strain CB15 has type IV pili and its flagellar motor switches direction of rotation to cause forward (cell body leading) and backward (cell body trailing behind its flagellum) swimming. CB15 Δpilin is a mutant of the wild-type strain that lacks pili, but it can also swim forward and backward much like the wild-type [33]. SB3860 is a double mutant that lacks pili and can only swim in the forward direction [34,35]. These three strains are illustrated in Figure 1. A close comparison of these three strains allows for determination of specific roles of pili and motor switching, if any, on motility. The goal of our study is to discern to what extent the motion of a uniflagellated bacterium, with or without pili, is affected by a well-defined, viscoelastic medium, and what features of the motility are changed in comparison with earlier findings on other species of flagellated bacteria [6][7][8]. The implications of the findings in this study are discussed along with details of the experimental results. Figure 1 Schematics of three strains of Caulobacter crescentus used: CB15 wild-type, CB15 Δpilin, and SB3860. The CB15 wild-type swarmer cell expresses pili (thin hairs) and the flagellar motor switches rotation directions to cause alternating forward and backward motion. The Δpilin cell expresses no pili, but the motor operates as the wild-type. The SB3860 (cheR138::Tn5 in YB375) is a double mutant, expressing no pili and moving exclusively forward.

Results and discussion
Our study was designed to test how motility of uniflagellated bacteria is affected by certain physical characteristics of the medium. Specifically, we chose polyethylene glycol and polyethylene oxide of selected molecular weights, mixing them into the cell medium in order to examine how changes in polymer size, concentration, and solution viscosity affect bacterial motility. We then compared changes in motility and viability of the three selected strains in a PEG/PYE medium in contrast to cells in glycerol/PYE medium. Finally, we delved into measurements of flagellum motor switching in order to detect subtle changes to its biochemical regulation, if any, caused by the test viscous agent.

Loss of motility attributable to apparent viscosity of polymer matrix
In a rich growth medium such as peptone yeast extract (PYE), C. crescentus usually spends around 30 minutes in the swarmer stage of its life cycle, after which it sheds its flagellum, thereby losing its motility [22]. Mixing PEG or PEO with young swarmer cells, we observed slight decreases in the percentage of motile cells. As a function of the polymer concentration, we found the decrease to be highly sensitive to the polymer size. Whereas 0.5% PEO 400000 caused loss of motility by over 50%, 10% PEG 4000 caused only about 10% loss of motility ( Figure 2A). This size sensitivity suggests strongly that rheological property of the polymer matrix might be the main cause for the loss of motility. Indeed, when plotting the same data as a function of the measured shear viscosity (listed on Table 1), we found the same gradual drop in motility, with the combined data in solutions of these three polymers collapsing to a single line ( Figure 2B).

Percentage of motile swarmer cells fell linearly as a function of viscosity
After being mixed with either a viscoelastic polymer (PEG or PEO) or a viscous medium such as glycerol, loss of motility was observed immediately. The percentage of motile cells decreased nearly linearly with viscosity ( Figure 3). As compared with solutions of equivalent viscosity, the glycerol solutions caused a much greater percentage of swarmer cells to become immobile than PEG did. The difference became more apparent in trials performed at higher viscosities. That the trend held across all three strains, CB15 wild-type, CB15 Δpilin, and SB3860 attests to the robustness of the observed property.
Among the three strains, the wild-type cells lost motility more steeply than the other two strains in both types of media as the viscosity increased. This difference suggests a specific role of pili on the loss of motility, which might be caused by direct interaction between the flagellum and pili that are known to be at the same pole of a C. crescentus swarmer cell. Such an interaction might be weaker and more transient at a lower viscosity, when the flagellum rotates with a higher speed. A faster rotating flagellum is expected to move past the surrounding pili faster, and perhaps less likely to get entangled, trapped, and stalled.
We observed no difference in the percentage of cells losing motility between the two Δpilin strains, with and without motor switching, in either the viscous or viscoelastic medium. This result suggests that the loss of motility, perhaps due to jamming or stalling of the motor, does not depend on motor switching mechanics. It also implies that, while increasing mechanical load on the flagellum can stall or jam the motor, the effect does not appear to be coupled to the switching behavior. Additional experiments were later designed to measure the motor switching behavior while varying the mechanical load, in order to further test this implication.

Significant cell death occurred at high glycerol levels but not in PEG/PEO media of comparable viscosity
We performed a specific test to see whether the loss of motility at high viscosity was reversible using both Δpilin strains. We first mixed swarmer cells with 48% glycerol by mass, and noted that within minutes all cells stopped moving. We then diluted the mixture 1:1 with cell medium, so that the cells were eventually in 24% glycerol with a much reduced viscosity of 1.8 cP. Despite the expectation that over 60% cells would be motile based on Figure 3 for both strains if they were exposed to only 24% glycerol, all the cells that received a shock of 48% glycerol for a few minutes and then were reduced to 24% glycerol remained non-motile over a 30-minute observation period. We conclude that the loss of motility due to elevated levels of glycerol is irreversible.
Fluorescence staining using DiBAC showed that a significant percentage of cells treated by 44% glycerol died within the minutes required to mix glycerol with the cells in the growth medium. Figure 4 shows that about 65% lost motility when mixed with 44% glycerol and over 70% of these non-motile cells became permeable to the dye and were practically dead. In contrast, among those cells found non-motile when mixed with 4% PEG 35000 (41% of total) or in PYE control (14% of total), only 23% and 18% of the non-motile cells were found dead, respectively, which might have occurred during the sample preparation such as the synchronization, mixing by pipetting, etc.
Previous work by others had shown that at an elevated level of~50% or higher, glycerol caused the death of E. coli, due to severe osmotic stress or dehydration [36]. On the other hand, it is a widely accepted practice to store bacterial specimens at −80°C in relatively low concentrations of~15-25% glycerol, in order to prevent ice crystal formation and rapid water loss during the freeze and thaw process [37]. Although we found no similar studies for C. crescentus, the findings for E. coli offers a crude guideline for the lethal level of glycerol [36,38], which appears consistent with the level we found for the total loss of motility and viability of C. crescentus (48%).
We also measured the osmolarity of 10% glycerol in comparison with that for 10% PEG 4000, 5% PEG 35000, and 5% PEO 400000. The results are listed in the Additional file 1: Table S1, confirming that glycerol produced much stronger osmotic pressure at comparable viscosity than that caused by introducing polymers into the cell medium. These additional measurements confirmed the mechanism detailed by earlier studies with glycerol, while also suggesting that adding inert polymers into the cell medium may be the better approach for the study of bacterial motility at elevated viscosity.
In conclusion, high percentage glycerol levels cause loss of motility and viability, likely attributable to hyperosmotic shock. In contrast, large PEG/PEO polymers appear to cause more moderate loss of motility by stalling the flagellar motor without killing the cells.

Polymer matrix attenuates the decrease of swimming speed
Here we switch gears to describe our findings on the influences of glycerol and polymer matrices on the swimming   speed of the cells that remain motile. A close comparison of these two types of media led to insights into the physical process. Implications of the features we observed are briefly discussed. Based on previous reports, C. crescentus swarmer cells undergo "forward-backward-flick" motion as the flagellar motor switches between clockwise and counterclockwise rotations [39]. On average, the speed of the backward motion is the same as that of the forward motion in spite of subtle differences between their trajectories. As viscosity was increased, we observed decreasing swimming speeds of C. crescentus ( Figure 5). The speeds of all three strains were very similar to each other in the viscoelastic solution containing PEG 35000 and the viscous, Newtonian solution containing glycerol. In glycerol, the speeds decreased inversely proportional to viscosity, with a fit power of −1.06 (R = 0.98). The inverse relationship suggests that the flagellar motor maintains a constant torque (proportional to speed times viscosity) as the viscosity of the medium varies. Indeed, the flagellar motor of C. crescentus has been previously shown to exert a constant torque at a range of rotation rates, from~50 to~300 revolutions per second, with the upper bound being the motor speed found during free swimming in water [15]. The new finding confirms that, at the high load regime, the flagellar motor maintains a constant torque regardless of its rotational rate, which decreases as the viscosity of the surrounding medium is increased [14,15,40]. The motor rotation is known to involve a flux of protons through the MotA/MotB stator complex [1,41,42]. Our finding of constant torque also implies that the viscous agent, including even the fairly small glycerol molecules, does not interfere with the proton-motive path of the MotA/MotB complexes, which would otherwise have impeded the proton flow and altered the motor torque in some dose-dependent fashion. Note that our study is conducted under the high load regime in that all MotA/MotB units are presumably coupled with the flagellum motor. This scenario is rather different from the low motor load regime, under which interesting findings have been made recently on the flagellar motor of E. coli, which adapts to an increasing load with stepwise deployment of additional units, thereby increasing torque [2,[43][44][45].
In PEG 35000, we found a more gradual decrease in swimming speed with increasing viscosity (fit power −0.45; R = 0.96). In other words, the polymer network of PEG allowed for markedly higher speed as compared to an equally viscous solution of glycerol. This experimental result suggests that the swimming speed driven by a rotating flagellum is enhanced by the elasticity of the polymer matrix. In fact, more pronounced enhancements on Figure 4 Fractions of dead, non-motile and motile cells under three medium conditions. Cells whose membrane became permeable to DiBAC were considered dead. The non-motile cells were those whose membrane was still intact, so that the cells were not labeled by DiBAC. Observations were made with SB3860 cells within minutes following the preparation as described under Materials and Methods. The fractions of motile cells were determined prior to the labeling experiments, with the results taken from Figure 3. The numbers of non-motile cells counted in the labeling experiments were 467 in PYE, 1207 in 4% PEG 35000, and 565 in 44% glycerol.
swimming by a viscoelastic medium were previously shown on several other species of bacteria [6][7][8]. Note those earlier works used methylcellulose and polyvinylpyrrolidone (PVP), which are much larger in size than the polymers we used in this study. When we performed similar experiments with C. crescentus using methylcellulose, we observed a peak in swimming speed at 0.05% (viscosity~1.4 cP; data not shown). The latter result is consistent with previous observations of other species of bacteria [6][7][8]. The comparison here suggests that the difference in speed-viscosity behavior between our measured speeds in PEG 35000 (no peak) and those previously published by others using methylcellulose and PVP is likely due to the difference in matrix molecular size rather than species-specificity. The extremely large sizes of methylcellulose and PVP (on the order of a million daltons) appear to have caused enough enhancements on thrust, over-compensating the expected decrease in swimming speed due to increased drag. As a result, a peak in swimming speed has been predicted based on hydrodynamic calculations [11,46]. Unfortunately, a rigorous comparison between the theoretical predictions and the experimental results is hampered by not knowing the actual size of the extremely high molecular weight polymers used in those experiments, and at the present time a lack of rheology data for those polymers at the low concentrations, which are necessary for bacteria to remain motile. In fact, our ongoing effort is designed to be conducive to more rigorous mechanical analysis using PEG and PEO, which cover a large range of available sizes.

Motor switching and stalling may be differentially regulated
We further explored whether a viscoelastic medium might affect the motor switching behavior by increasing the mechanical load on the flagellum. We measured the average switching frequency of wild-type and Δpilin cells in the 4% PEG medium in comparison with that of PYE. A set of four movie clips via links in the Additional files 2, 3, 4 and 5 illustrates switching events that occurred for both wildtype and Δpilin cells in PYE and 4% PEG 35000 media. Figure 6 shows overlays of selected movie frames where cells appear as bright spots and locations of switches are marked. The high frame rate of the recorded movies ensured sufficiently high time resolution of~10 ms for our measurements (See details in Materials and Methods).
A close comparison of switching behavior of both wildtype and Δpilin strains was made, with results shown in Figure 7 and Table 2. We found no significant difference between the cells with and without pili (Figure 7). There is a small increase in switching frequency for both cell types in 4% PEG 35000 medium, but the visual displacement in peak position is much smaller than the natural spread of the measured intervals. A Student T-test performed in comparison with the average intervals confirms no significant difference. Note also the large standard deviations of the switching intervals under all 4 conditions ( Table 2).
The simplest yet an important conclusion of this comparison is that there was insignificant difference among all 4 measured conditions. It was not possible to perform the same measurements for cells in glycerol-PYE medium of comparable viscosity to that of 4% PEG (4.6 cP). This would have required 48% glycerol, at which concentration all cells totally lost their motility.
Our findings from the switching interval measurements strongly suggest that the switching and stalling of the flagellar motor are differentially regulated. The gradual loss of motility in either a viscous or viscoelastic medium does not depend on whether or not the motor switches ( Figure 2); the motor switching frequency varies little with the addition of PEG 35000 up to 4%, which increases the apparent viscosity 5 fold. The loss of motility appears to be a sudden stall or jam, either due to injury caused by hyperosmotic shock at elevated levels of glycerol, or triggered by some mechanical failure under a high mechanical load or stress. Once the stalling occurs, the damage to the motor appears permanent, evidenced by the irrecoverable loss of motor function, and subsequent loss of the cell's viability. We note that under the natural environment of relatively low fluid viscosity, the C. crescentus flagellar motor frequently switches its rotation direction with very short or even non-detectable stall time. In contrast, there are also species such as R. sphaeroides for which the motor can pause as part of its natural cycle of motion [47]. We observed a few instances when a Caulobacter cell paused and then resumed motility.

Conclusions
This study was designed to assess and differentiate effects of solution viscosity and viscoelasticity on the swimming behavior of C. crescentus as a simple microswimmer with one helical flagellum. The results clearly show quantitative differences between a polymer matrix as a viscoelastic medium and a Newtonian fluid with elevated viscosity. In the Newtonian fluid, we found the swimming speed to scale inversely proportional to viscosity, as expected based on hydrodynamic theory for constant motor torque. In the test polymer network, we found the swimming speed to fall more gradually with viscosity, as v~η -0. 45 . This result suggests that the flagellated micro-swimmer produces a larger thrust at an increased polymer concentration, which partially offsets increased drag on the cell body. We were unable to quantitatively account for the measured results based on fluid mechanical calculations, due to lack of knowledge of detailed rheological properties of the PEG medium. Specifically, the apparent viscosity measurements performed in this study do not yield values of the storage and loss moduli as functions of the frequency of perturbance exerted by the rotating flagellum. Thus, additional characterization of the polymer matrix is required to better define the mechanical effects of the chosen media on the bacterial swimming speed. Figure 6 Overlays of selected frames at 0.17 s intervals out of a 3-second movie, to illustrate switches during the motion of Δpilin (left panels) and wild-type (right panels) cells in PYE (top panels) and 4% PEG 35000 (bottom panels). The faint grey lines following the swimming trajectories were produced with darkened overlay of all frames over the 3-second movies at 125 frames/second. Arrows next to the trajectories were drawn to point out the directions of motion, with switches indicated by grey circles. The scale bar applies to all 4 images.
By performing experiments using three selected strains, we were able to distinguish common behavior robustly accounted for by mechanical properties of the media with some notable exceptions, which offer insights into the functions of the flagella motor. The quantitative dependence of swimming speed on viscosity or concentration of polymers added is practically identical among all three strains. In both media, there is a significant difference in fraction of immobilized cells observed between the wild-type cells and the two strains lacking pili. Additionally, we saw nearly no effect of a viscoelastic medium on the switching frequency of the flagellar motor of C. crescentus. The results in this report call for further study on the control of the flagellar motor, as well as more detailed mechanical analysis. The latter requires high-speed imaging of the flagellum in order to determine its relative orientation to the cell body, as well as the hook that connects them. On the other hand, a more comprehensive characterization of the frequency dependent rheological properties of the polymer matrix is also required at the range most relevant to bacterial motility.
We conclude by suggesting that our study of bacterial motility in complex fluids using C. crescentus as a model system may spark interest in similar experiments on other flagellated bacteria in diverse environments, particularly those that find viscoelastic media their natural habitat.

Bacterial strains
Caulobacter crescentus strains CB15 wt, CB15 Δpilin (YB375) [33], and SB3860 were used. SB3860 is cheR138:: Tn5 in YB375, ie., a mutant of CB15 Δpilin. It was kindly generated by Bert Ely, and was used in our previous studies [34,35]. As described previously [33,48], the strains were grown in peptone yeast extract (PYE) medium [23] and synchronized with the plate release method [49], modified recently [22,48]. The synchronization procedure yielded swarmer cells in fresh PYE that were within 5 minutes of division. The synchronized swarmer cells were immediately used in the experiments. Video recordings were terminated before the swarmer cells reached the age of 30 minutes, ensuring that properties observed were not complicated by the onset of differentiation towards the non-motile stage.

Viscometry and osmolarity measurements
We used polyethylene glycol of average sizes of 35,000 and 4000 daltons (PEG 35000 and PEG 4000, respectively; Sigma-Aldrich) and polyethylene oxide of 400,000 daltons (PEO 400000, Sigma-Aldrich). PEG 35000 has a radius of gyration of 8.6 nm [16,50], and the polymer becomes entangled in solutions at about 2% by weight or mass, estimated based on the Rouse model of polymer chains [51,52]. The corresponding numbers for PEG 4000 are 2.9 nm and 6% and for PEO 400000 are 29 nm and 0.6%, respectively. Solutions of PEG 35000 or glycerol (Fisher Scientific) in PYE medium at room temperature were prepared to vary their viscosity up to 5-7 times to water. The PEG(PEO)/PYE and glycerol/PYE mixtures were made by first weighing a highly viscous stock solution such as 40% PEG 35000 (in PYE), 5% PEO 400000 (in PYE), or 100% glycerol, using an analytical balance, Figure 7 Histograms of intervals between motor switches. The intervals were measured for wild-type (wt, circles) and Δpilin (deltaP, triangles) cells in the standard PYE medium (solid symbols) and in the PYE medium containing 4% PEG 35000 (hollow symbols). The data show slight shift of switch intervals to lower values due to addition of PEG 35000. More details of these data are tabulated in Table 2. We used the calibrated viscometer to measure the shear viscosity of the cell medium containing PEG or PEO. Solutions containing PEG 4000 over the 0-10% range yielded viscosities up to 3.0 cP. Solutions containing PEG 35000 over the 0-5% range yielded viscosities up to 6.5 cP. Solutions containing PEO 400000 over the 0-1% range yielded viscosities up to~15 cP. We searched the literature and found these values to be consistent with values measured for PEG of several other molecular sizes, considering the known trend of a sharp increase with molecular weight at fixed mass concentration [50]. Our measured values are tabulated and plotted in Additional file 1.
We measured the osmolarity of 10% glycerol, the PYE growth medium and selected PEG/PEO solutions, using a commercial osmometer based on freeze point depression detection (Osmette II, Precision Systems, Model 5005, Natick, MA).

Measurements of motility
An aliquot of synchronized cells in PYE was mixed with a viscous medium in a 1 mL centrifuge tube, by gently pipetting for 2-5 minutes, to uniformity. The final mixtures contained up to 10% of PEG 4000, 5% of PEG 35000, 0.5% of PEO 400000, or 48% of glycerol, all by mass. A 115-150 μL aliquot of bacteria-viscous agent mixture was then placed within a chamber formed by a 12.7 mm-diameter rubber O-ring, glued on top of a microscope slide using epoxy. The bacteria sample was made with sufficient depth (~1 mm) in order to collect data about swimming behavior far from the fluid boundary. A cover slip was placed on top of the O-ring filled with the sample to form a seal and allow for convenient observation using an upright microscope. Movies for counting motile versus non-motile cells were recorded between 2 and 5 minutes after mixing unless longer times were specified.
Images and videos were taken using a Nikon Eclipse E800 upright optical microscope with a mounted Cool-Snap CCD camera (Photometrics, Tucson, AZ) and the software MetaMorph (Universal Imaging Corp., 2002). In order to capture C. crescentus during the swarmer stage of its life cycle, all observations were made on swarmer cells within 30 minutes in age.
To collect videos for motility detection, we used a long working distance 40x phase-contrast objective with a 0.05 s exposure time and a low capture rate of 6.7 frames/second. Under the 40x objective magnification, individual swarmer cells were discernable as opposed to cell aggregates or pre-divisional cells, which were both excluded from the counts. In contrast to swimming cells, which moved several microns per frame, non-motile cells displayed only diffusive motion and moved much less than 1 μm/s. Since the difference between them was over tenfold, the motile and non-motile cells were distinguished by eye. Percentages of immobilized cells were obtained by counting single swarmer cells that did not swim compared with those that were swimming in the recorded short videos.
Two methods were used to yield appropriate error bars. Method one applies to the percent motile data for strain 3860 in PEG 4000 and PEO 400000. Ten videos of ten frames each were taken within two minutes. Within each video, a few dozen cells were counted, out of which one value of percent motile was obtained. These ten values were averaged to yield the percentage motile along with an error bar. Method two applies to the percent motile data of all three strains in glycerol and PEG 35000. It extends beyond method one as similar measurements under these conditions were repeated two more rounds. The final percent motile data were shown as the average from the three rounds of measurements and the standard error from the three values. The errors are smaller by the second method since three times as many cells were counted under each condition of those measurements.

Measurements of swimming speed
For measurements of swimming speed, samples were prepared using the same rubber o-ring as for the motility assay. A sample was typically placed on the microscope stage for 5-7 minutes after mixing before recording to ensure the decay of fluid flow, which was the main source of error. A 20x objective was used with the dark-field setting of the microscope to take videos with 0.1s exposure time at 6.7 frames/second for 20-50 frames. The swimming speeds were obtained by analyzing the dark field videos using a custom-written MATLAB (Mathworks, Inc., Natick, MA) program. The program selects for swimming trajectories with positions discernable over ≥10 frames and without sharp changes of direction (threshold set at 1 radian/second) between frames in order to exclude measuring speed during motor switching events. Instantaneous speed was determined based on the spacing between consecutive cell body positions. The average speed over a short trajectory of each cell (during either forward or backward motion, as the difference in speed is negligible) was calculated for dozens or hundreds of cells under each condition. Due to the natural spread of swimming speed among individual cells, our data were plotted with standard deviation, which does not depend on how many cells were measured under each condition.

Measurements of cell viability
Following an established method [53], we used a fluorescent dye called DiBAC(4)3, (bis-[1,3-dibutylbarbiturate] trimethine oxonol) (Sigma-Aldrich, Inc.), which enters depolarized cells and exhibits enhanced fluorescence. Thus the DiBAC dye was chosen to label those C. crescentus cells that had lost their membrane integrity and were considered dead. A stock solution of 1 mg/mL DiBAC was dissolved in 1:1 ethanol:water mixture and diluted 1:1000 into synchronized swarmer cells in PYE (control), PYE containing 44% glycerol, or PYE containing 4% PEG 35000. Thin samples were prepared the same way as described below for the motor switching experiment. Each sample was imaged using both phase contrast and fluorescence microscopy. A long exposure time (0.5 s) was used so that motile cells appeared as faint streaks under phase contrast. The number of nonmotile cells appearing in each image was counted and the fraction of non-motile cells that were dead (i.e. appeared under both phase contrast and fluorescence) was calculated.

Measurements of motor switching
For measurements of motor switching, thinner samples were found to yield better images as the cells were prevented from moving out of the focal plane. A 5 μL sample was pressed between a microscope slide and a coverslip, sealed with a layer of vacuum grease around the edges. The sample thickness of such preparations was on the order of 10 μm.
Videos were taken using a Photron FASTCAM-PCI R2 high-speed camera mounted to a Nikon Eclipse TE2000-U inverted optical microscope. Videos were recorded using a 20x phase-contrast objective with a frame rate of 125 frames/second. The time interval between motor switches was measured by visually identifying the frames at which a particular cell underwent a reversal in its direction of travel. The time interval between two consecutive switching events was then calculated based on the known video frame rate. The high frame rate ensured that the measurement error was within 10 milliseconds, far shorter than the intervals between motor switches, which typically occur on the order of a second.


Two Types of Antibodies Are Induced by Vaccination with A/California/2009pdm Virus: Binding near the Sialic Acid-Binding Pocket and Neutralizing Both H1N1 and H5N1 Viruses

Many people have a history of catching the flu several times during childhood but no additional flu in adulthood, even without vaccination. We analyzed the total repertoire of antibodies (Abs) against influenza A group 1 viruses induced in such a flu-resistant person after vaccination with 2009 H1N1 pandemic influenza virus. They were classified into two types, with no exceptions. The first type, the products of B cells newly induced through vaccination, binds near the sialic acid-binding pocket. The second type, the products of long-lived memory B cells established before vaccination, utilizes the 1-69 VH gene, binds to the stem of HA, and neutralizes both H1N1 and H5N1 viruses with few exceptions. These observations indicate that the sialic acid-binding pocket and its surrounding region are immunogenically very potent and majority of the B cells whose growth is newly induced by vaccination produce Abs that recognize these regions. However, they play a role in protection against influenza virus infection for a short period since variant viruses that have acquired resistance to these Abs become dominant. On the other hand, although the stem of HA is immunogenically not potent, the second type of B cells eventually becomes dominant. Thus, a selection system should function in forming the repertoire of long-lived memory B cells and the stability of the epitope would greatly affect the fate of the memory cells. Acquisition of the ability to produce Abs that bind to the stable epitope could be a major factor of flu resistance.

Introduction
Influenza is an infectious disease of the respiratory tract that affects millions of people every year. Since antibodies (Abs) play important roles in protection against influenza virus, preventive vaccination has been one of the most efficient measures of influenza control. Hemagglutinin (HA), which is the main target for virus-neutralizing Abs, mediates virus entry into cells at two steps [1]. First, HA binds to the cell receptor, sialic acid. After internalization of viruses by endocytosis, HA undergoes a drastic conformational change induced by low pH. Neutralizing Abs have one of the following activities: prevention of the binding reaction between HA and sialic acid [2] and prevention of low-pH-induced conformational change of HA [3]. The former binds to the site near the sialic acid-binding pocket on the globular head in HA1, and the latter binds to the stem region formed mainly by HA2. Since the dominant immune response is the first type and mutations can be easily introduced into the target sites without losing the receptor-binding activity, variant viruses that have acquired resistance to these Abs become dominant and cause annual epidemics. Historically, it was long believed that all effective neutralizing Abs are the first type; therefore, vaccine strains should be changed almost every year to remain effective. As long as the vaccine strain is a good match to the circulating virus, vaccination is effective for preventing virus infection.
The mode of response against influenza virus infection is very heterogeneous among the human population. For example, many people who have experienced several influenza infections in their childhood have not experienced additional influenza infections, even without vaccination. There has been no report clearly showing what kinds of ability such flu-resistant people obtained through repetitive influenza infections in their childhood. In 2009, a swine-origin H1N1 influenza virus (S-OIV) emerged and spread rapidly among the human population, resulting in its classification as the first pandemic of the 21st century [4]. It has been generally believed that humans lack immunity to the newly appearing influenza virus at the outbreak of a pandemic because they are naive to the virus. In the case of S-OIV, however, long-lived memory B cells that produce broadly neutralizing Abs not only against seasonal H1N1 viruses but also S-OIV are found in many elderly individuals [5][6][7].
To analyze the total repertoire of neutralizing Abs against influenza viruses in humans, we developed the following exper-imental strategy in our previous study [8]. A large number of B lymphocytes are collected by apheresis from a donor, and a huge Ab library is constructed by using phage-display technology. The library is screened with formalin-treated virus particles. The clones that have both binding activity and neutralizing activity to the virus are isolated and their strain specificity is extensively characterized. Although only B lymphocytes that are circulating in peripheral blood are analyzed in our experimental system, the results appeared to represent the total repertoire of neutralizing Abs formed in the donor's body [8]. In the present study, we applied this strategy to the analyses of Abs that are present in the human body before and after vaccination with a newly appearing kind of virus. Since S-IOV newly emerged, the results should reflect only the effects of vaccination without the effects of a natural infection with living S-IOV.

Viruses
The following influenza viruses were used in this study.

Ethics statement
Ethical approval was granted by the Research Ethics Committee of Fujita Health University. Signed informed consent was obtained from the blood donor.

Construction of Ab library
Phage Ab libraries were constructed as described previously [8]. Briefly, mononuclear cells from a donor born in 1947 were collected by apheresis from the equivalent of 3 litters of blood before and after vaccination. The cells included 8.0610 8 B lymphocytes (before vaccination) and 1.2610 9 B lymphocytes (after vaccination). Large combinatorial Ab libraries were constructed by using the phage-display method as described previously [9]. The size of the libraries were: before vaccination, 1.6610 9 clones for heavy (H) chain, 2.0610 9 clones for light (L) chain and 1.4610 10 clones for Fab; after vaccination, 3.2610 9 clones for H chain, 1.3610 9 clones for L chain and 2.6610 10 clones for Fab.

Screening of the library
Phages bound to virus particles were selected by a panning method as described previously [10]. In brief, formalin-treated virus particles of Cal09 or Bri07 strains were used as antigens (Ags) in the screenings. After two and three pannings, E. coli (DH12S) cells were infected with the eluted phages and spread onto the LB plates containing 100 mg/ml ampicillin and 0.2% glucose. E. coli colonies harboring phagemid were picked up and grown in 26YT medium containing 100 mg/ml ampicillin, 0.05% glucose and 1 mM isopropyl-b-D-thiogalactopyranoside at 30uC overnight. During growth of E. coli, the Fab-cp3 form of Ab was secreted into the medium [11]. The culture supernatants containing Fab-cp3 molecules were subjected to enzyme-linked immunosorbent assay (ELISA) against H1N1 virus used as Ag in the screening and H3N2 virus. Clones that bound only to H1 were selected and subjected to further analyses.

ELISA
Formalin-treated virus particles were coated onto 96 well Maxisorp immunoplates (Nunc), and Fab-cp3 Ab in the supernatant of E. coli culture was added to each well. After incubation with rabbit anti-cp3 Ab (MBL), the wells were further incubated with peroxidase-conjugated goat anti-rabbit IgG (H+L chain; MBL). Then, HRP substrate (OPD; Wako) was added to each well, and the color of the sample was developed. After stopping the peroxidase reaction by adding H 2 SO 4 , the absorbance of the sample at 492 nm was measured.

Sequence analysis
The nucleotide sequences of V H fragments of isolated Ab clones were determined by using GenomeLab Dye Terminator Cycle Sequencing with Quick Start Kit (Beckman Coulter) and a CEQ2000 DNA Analysis System (Beckman Coulter). The T7ETZ (59-TAATACGACTCACTATAGGG-39) was used as the V H sequencing primer.

Virus neutralization test
For measurement of virus neutralizing activity, a focus reduction assay [12] was performed by using the single cycling (VN) or multiple cycling (M-VN) method. Two hundred or 500 mg/ml of Fab-PP Abs (P denotes a single Fc-binding domain of protein A) or two-fold serial dilutions of serum were mixed with an equal volume of 100 FFU of influenza virus and applied to MDCK cells in a 96-well plate. In the VN method, after incubation with the mixture, the cells were washed with serum-free MEM and cultured in MEM containing 0.4% BSA at 37uC for 15 h. In the M-VN method, after incubation, MEM containing 0.4% BSA, 5 mg/ml of acetylated trypsin, and 0.5% methyl cellulose of equal volume to the mixture was further added to the cells without removing the mixture and the cells were incubated at 37uC for 28 h. Then, the cells were fixed with ethanol and stained with PAP (peroxidase and anti-peroxidase) complex. The number of foci containing one and more cells (VN method) or four and more cells (M-VN method) was counted. The results were indicated as the focus reduction rate (%) for Fab-PP Ab or the reciprocal of the highest dilution of serum to show 50% focus reduction rate for serum.

Hemagglutination inhibition (HI) assay
The HI test was performed as described previously [12]. In brief, serial dilutions of 160 mg/ml of purified Fab-PP or donor's serum in PBS were prepared. Serial dilutions of Fab-PP or serum were preincubated with 4 HA units of virus per well. Guinea pig red blood cells (0.75%) in PBS were added to each well, and the plate was incubated at room temperature for 30-60 min. The results were shown as the lowest concentration (mg/ml) of Fab-PP Ab or the reciprocal of the highest dilution of serum to inhibit hemagglutination.

Competition ELISA
Competition ELISA was performed by using the Fab-PP form of Ab [13] or C179 [3] for detection of the binding activity to virus particles and the Fab-cp3 form of Ab as a competitor. Fab-cp3 molecules in the supernatant of E. coli culture were concentrated 20-fold before use. Formalin-inactivated virus particles were coated onto a 96 well Maxisorp immunoplate. A total of 50 ml of Fab-PP at an optimized concentration was mixed with 50 ml of 20-fold concentrated Fab-cp3 and the mixture was added to a virus-coated well. Then, peroxidase-conjugated rabbit anti-streptavidin Ab was added to each well as a secondary Ab. When C179 at the final concentration of 0.25 mg/ml was used for detection of the binding activity to virus strain, each well was incubated with peroxidase-conjugated goat anti-mouse IgG (H+L chain; MBL) as a secondary Ab. Then, HRP substrate (OPD; Wako) was added to each well, and the color of the sample was developed. After stopping the peroxidase reaction by adding H 2 SO 4 , the absorbance at 492 nm was measured.
Preparation of mAb specifically bound to 1-69Abs Five kinds of Fab-PP form of 1-69Ab, F081-007, F083-103, F083-115, F083-305, and F083-311 were purified and injected into the foot-pad of Balb/c mice with Freund's complete adjuvant once and 11 days later without adjuvant once more. Three days later after the second injection, the lymphocytes were isolated from the inguinal lymph node and fused with the myeloma line P3-X63AG8.653. After cloning of hybridomas, the culture media were screened using the above 1-69Abs and various kinds of Abs that do not utilize 1-69 V H gene by ELISA. The clones that bind to multiple kinds of 1-69Ab but do not bind to any of non 1-69Abs were selected. This part of the work was performed by MAB Institute Inc. (Sapporo, Japan).

Determination of the serum concentration of IgG that uses the V H 1-69 germline gene
A human IgG ELISA Quantitation set (Bethyl Laboratories, Inc.) was used, with slight modification. K1-18 Ab for detection of IgG using V H 1-69 germline gene and affinity-purified Human IgG Coating Antibody for standard curve were coated onto a 96well Maxisorp immuonoplate. Serum or human reference serum (for standard curve) was added to the assigned well. After incubation with HRP-conjugated Human IgG Detection Antibody, TMB substrate was added to each well. The peroxidase reaction was stopped by adding H 2 SO 4 , and the absorbance of sample at 450 nm was measured. The concentration of IgG bound to K1-18 Ab was calculated from the standard curve of human reference serum.

Virus-neutralizing activity of serum in the presence of K1-18 Ab
The virus neutralization test described above was modified. In brief, serum treated with receptor destroying enzyme (RDE) was diluted at 1:10 or 1:20 in serum-free medium and mixed with an equal volume of 800 or 1,600 mg/ml of K1-18 Ab. After incubation, two-fold serial dilutions of the mixture were mixed with an equal volume of 100 FFU of influenza virus and applied to MDCK cells in a 96-well plate. After incubation, the cells were fixed with ethanol and stained with PAP (peroxidase and antiperoxidase) complex. The reciprocal of the highest dilution of serum to show a 50% focus reduction rate was indicated as virus neutralizing activity.
Binding activity of K1-18 Ab to 1-69Abs Ten mg/ml of Fab-PP form of 1-69Ab was coated onto 96 well Maxisorp immunoplates (Nunc), and K1-18 Ab was added to each well. After incubation with peroxidase-conjugated goat anti-mouse IgG (H+L chain, MBL), HRP substrate (OPD; Wako) was added to each well. After stopping the peroxidase reaction by adding H 2 SO 4 , the absorbance of the sample at 492 nm was measured.

Binding activity of Abs to virus particles under the presence of K1-18 Ab
Formalin-inactivated virus particles (Bri07) were coated onto a 96 well Maxisorp immunoplate. Fab-cp3 molecule that was diluted from 20-hold concentrated solution at an optimized concentration was mixed with an equal volume of 200 or 400 mg/ml of K1-18 Ab and incubated at 37uC for 1 h. The mixture was added to virus coated well and the wells were incubated at 37uC for 1 h. After incubation with rabbit anti-cp3 Ab (MBL), the wells were further incubated with peroxidase-conjugated goat anti-rabbit IgG (H+L chain; MBL). Then, HRP substrate (OPD; Wako) was added to each well, and the color of the sample was developed. After stopping the peroxidase reaction by adding H 2 SO 4 , the absorbance of the sample at 492 nm was measured.

Nucleotide sequence accession number
The nucleotide sequences of the heavy and light chains of isolated Abs have been deposited in the DDBJ database (accession numbers AB826498 to AB826560 for the heavy chains and AB845357-AB845391 for the light chains).

Two types of virus-neutralizing Abs with different characteristics
We examined the total repertoire of Abs induced by vaccination with S-IOV. The blood donor in this study was born in 1947 and was infected with influenza several times in his childhood (possibly by H1N1 and H2N2) and in 1968 (probably by H3N2). For 41 years afterwards, he did not contract influenza, and he was never vaccinated against influenza. The schedule of vaccination and blood collection is shown in Figure 1. The vaccination and blood collection were performed during the period from late October to mid-December 2009, and the examinee did not have an opportunity to be naturally infected with S-IOV. By using B lymphocytes collected before and after vaccination, two large Ab libraries were constructed and subjected to screenings by panning with pandemic H1N1 (Cal09) and seasonal H1N1 (Bri07) virus particles. After the second and the third rounds of panning with the viruses, 120 clones were isolated. The clones that bound to the H1N1 virus particle that had been used for screenings were further analyzed. The clones that bound to both H1N1 and H3N2 with equal strength were excluded, since it is likely that they were anti-NP Abs [8]. The HA-binding activity of respective clones was further confirmed by Western blot of virus proteins used as Ags in the screenings. Among the 240 clones isolated in respective screenings, the number of clones that were judged to be anti-HA Abs is as follows: screening 1 (after vaccination, with pandemic virus), 105 clones; screening 2 (before vaccination, with pandemic virus), 3 clones; screening 3 (after vaccination, with seasonal virus), 58 clones; and screening 4 (before vaccination, with seasonal virus), 16 clones. V H nucleotide sequences of all these clones were determined. Comparison of the amino acid sequences revealed that these 182 clones were composed of 96 unique monoclonal Abs (mAbs). Based on sequence similarities of V H fragments, the 96 clones were classified into 63 groups. The amino acid sequence of the V H fragment and the nucleotide sequence of complementarity-determining region 3 (CDR3) of all the clones are available in Figures S1 and S2.
The HA binding activity, hemagglutination inhibition (HI) activity, and the virus-neutralizing activity [12] of representative clones of the 63 groups were examined. As shown in Figure 2, all of the representative clones were classified into two types with no exceptions. Clones classified as the first type bind only to the pandemic H1N1 and not to the seasonal H1N1. All of these clones show HI activity and were isolated only from the screening 1. Clones classified as the second type bind not only to the pandemic H1N1 but also to all of the seasonal H1N1s. While they do not show HI activity, most of them neutralize not only H1N1 viruses but also H5N1 virus. Judging from the low frequency of mutations, such as 0% to 5%, the majority of the first type of clones should be the products of B cells that have been newly induced through vaccination. On the other hand, all of the second type of clones (except for a few) should correspond to the products of long-lived memory B cells that were established before vaccination, judging from the high frequency of mutations, such as 10% to 15%. Furthermore, they all utilized the 1-69 V H gene, with the exception of three clones.

Newly appearing Abs after vaccination
Since virtually all the first-type Abs showed HI activity, it is likely that their epitopes are located in the region surrounding the sialic acid-binding pocket. To systematically examine the relative position of the epitopes recognized by these clones, we adopted the competition method that we used in a previous study [14]. While respective mAbs are initially prepared as the Fab-cp3 form after the screening of libraries, they can be easily changed to the Fab-PP form (P denotes a single Fc-binding domain of protein A) in our vector construct [13]. If the epitope recognized by clone A is overlapped with that by clone B, the binding of the Fab-PP form of clone A to HA is largely disturbed by the presence of a large amount of Fab-cp3 form of clone B. Based on this principle, the competition study was performed by using 17 clones selected from the first-type Abs listed in Figure 2. As shown in Figure 3, 14 of the 17 clones competed well against one another for the binding to HA. In the case of three clones, F082-317, F082-254 and F082-022, the degree of disturbance for the binding was low. These observations are consistent with other data. Only F082-022 showed very low HI activity (titer 160 mg/ml). F082-254 binds to HA of not only pandemic virus but also seasonal virus and neutralizes both viruses. Furthermore, a high frequency of mutation, such as 18%, was observed in this clone. In the case of F082-317, the binding activity to HA is high but the neutralizing activity is relatively low.
Based on these observations, we concluded that the sialic acidbinding pocket and its surrounding region on HA are immunogenically very potent and that virtually all of the B cells whose growth is newly induced and expanded by vaccination produce Abs that recognize these regions. We also concluded that when formalin-treated virus particles that are not alive are used as vaccine, B cells producing Abs that are able to bind to HA but not able to neutralize virus are not induced at a substantial level. The second conclusion suggests that the non-neutralizing epitope on HA, even if it exists, is immunologically impotent.

Abs encoded by long-lived memory B cells
While the second type of clones is classified into 32 groups, they all utilized the 1-69 V H gene, with the exception of three clones. Furthermore, the majority of these Abs were able to neutralize not only all H1N1 viruses but also the H5N1 virus. These observations suggest that the epitopes recognized by these clones should be located near the epitope recognized by C179, as shown by several groups [15,16]. We examined whether the binding of C179 to HA is disturbed by a large excess of the Fab-cp3 form of these clones. As shown in Figure 4, all of the clones including the three that did not utilize the 1-69 V H gene disturbed the binding of C179 to HA. Thus, the second type of clones should bind to the membraneproximal stem of HA in the same or similar way as other Abs using the 1-69 V H gene (1-69Abs), such as CR6261 and F10, which have already been described by other groups [15,16]. Figure 4 also indicates that four clones, F081-268, F082-243, F082-237, and F083-373, showed weaker disturbance activity than the others. The data shown in Figure 2 indicate that F081-268 had strong HA-binding activity, but the neutralizing activity was weak. The other three clones showed relatively weak binding and neutralizing activities. Thus, while all of the epitopes recognized by the second type of Abs are not exactly the same as that recognized by C179, we did not find any clone that binds to an epitope totally different from that recognized by C179. This result suggests that this epitope has been shared and stably kept among group 1 viruses. Even if the immunogenicity of this epitope is weak, once humans acquire B cells that produce Abs that recognize this epitope, the B cells could remain in the body as memory cells for a long period and become major players against group 1 viruses. Interestingly, one clone (F083-115) that utilized the 1-69 V H gene is able to neutralize even H3N2 virus.

Presence of Abs secreted into the serum
Two biological activities, HI activity and virus-neutralizing activity, against three viral strains, H1N1 pandemic virus, seasonal H1N1 virus and H5N1 virus, were measured in eight samples of sera collected from the examinee on different days, as shown in Figure 1. As indicated in Table 1, in the case of HI, the activity against seasonal H1N1 and H5N1 viruses was not detected in any of the sera. HI activity against H1N1 pandemic virus started to appear 2 weeks after the first vaccination. In the case of virusneutralizing activity measured by standard focus reduction assay, the activity against H1N1 pandemic virus already started to increase 1 week after vaccination and reached a plateau 2 weeks after vaccination. Against seasonal H1N1 virus, virus-neutralizing activity was detected even before vaccination and increased 1 week after vaccination. Against H5N1 virus, virus-neutralizing activity Figure 2. Activities of representative clones classified into 63 groups. The first three columns contain the numbers of clones isolated by the screenings. Germline genes were identified by comparing the amino acid sequence of V H of the representative clone with the sequences of all of the germline V H genes, and the identity (%) is indicated. The amino acid sequence of CDR3 is shown. The binding activity to four H1N1 (NC99, SI06, Bri07 and Cal09) and one H3N2 virus particles was examined by enzyme-linked immunosorbent assay (ELISA). Absorbance at 492 nm is shown as follows: $1.00 (red), 0.50-0.99 (orange), and 0.10-0.49 (yellow). The virus neutralizing activity of 100 or 250 mg/ml Fab-PP Ab against H1N1, H5N1, and H3N2 viruses was examined by the focus reduction test. The reduction rate is shown as a percentage as follows: $60% (dark blue), 40%-60% (blue), and 20%-40% (light blue). The HI activity of Fab-PP Ab was measured by using two H1N1 (Bri07 and Cal09) viruses. The lowest concentration (mg/ml) of Fab-PP Abs to inhibit hemagglutination is shown. doi:10.1371/journal.pone.0087305.g002 was not detected by the standard focus-reduction assay [12] but was detected by a more sensitive method that is described in Materials and Methods.
To prove that 1-69Abs are really responsible for neutralizing H1N1 pandemic virus, seasonal H1N1 virus and H5N1 virus, we prepared mAb that can specifically bind to 1-69Abs, that is, antiidiotypic Ab. We expected that the region including two amino acids, isoleucine at the 53rd residue and phenylalanine at the 54th residue, in the V H domain should be immunogenic in mice since the presence of two hydrophobic residues in CDR2 is very unique and found only in 1-69Ab of humans [17]. Furthermore, we presumed that the anti-idiotypic Ab may inhibit the binding of 1-69Ab to HA since these two amino acids are directly involved in the Ab/HA interaction [15,16]. We successfully isolated mAb K1-18 that can bind to around 80% of 1-69Abs listed in Figure 2 and can inhibit the binding of a half of the 1-69Abs to HA as indicated in Figure 5A and B.
By using K1-18 as a probe, the amount of the IgG form of 1-69Ab in serum was measured. The results indicated that it was present at a concentration of 4.58 mg/ml in the serum collected on October 30 (before vaccination) and increased after vaccination to an concentration of 11.24 mg/ml in the serum collected on December 14. Virus-neutralizing activity against pandemic and seasonal H1N1 viruses was measured in the presence of K1-18. In the case of H1N1 pandemic virus, K1-18 definitely inhibited the neutralizing activity, as indicated in Table 2. Against seasonal H1N1 virus, the inhibition was clearly observed, although not perfectly. Thus, we concluded that 1-69Abs were really present in Figure 3. Competitive inhibition of binding to HA among the Abs newly induced by vaccination. The binding activity of Fab-PP Ab (indicated at upper side) to Cal09 virus particles was measured by ELISA under the presence of a 10-times greater concentration of Fab-cp3 Ab (indicated at the left side). F008-009 and F033-367 are not anti-HA Abs and were used as controls. The binding inhibition was calculated as follows: the absorbance value under the presence of F008-009cp3 was used as 100% binding, and the degree of reduction in the absorbance value under the presence of Fab-cp3 of Ab was measured and shown as percent inhibition. Percent inhibition is shown as follows: $70% (white), 50%-70% (blue), 0%-50% (grey). The experiment was performed at least three times in duplicate. doi:10.1371/journal.pone.0087305.g003 the sera and functioned in the neutralization of H1N1 viruses. Furthermore, the data in Table 2 suggested that when two types of functionally different Abs co-exist, the first type prevents HA/ receptor interaction and the second type prevents low-pH-induced conformational change of HA, and the virus-neutralizing activity synergistically increases.

Discussion
Historically, it has been believed that vaccines remain the most reliable method to control seasonal epidemics of influenza. However, the mode of response against influenza virus infection is very heterogeneous among the human population. Since we experience an outbreak of flu almost every year in Japan, most of the people may easily have opportunities to be infected by influenza viruses. Nevertheless, there are many people who appear to be resistant to the seasonal epidemics without vaccination. On the other hand, there are many people who are vaccinated almost every year but contract influenza quite often. In the present study we analyzed the repertoire of neutralizing Abs against H1N1 viruses in a flu-resistant person. We also tried to analyze the effects of vaccination with formalin-treated virus particles on the Ab repertoire under the conditions without effects of natural infection with live viruses. This experiment can be performed only at the outbreak of a pandemic. Since people are naive to the pandemic virus, we can perfectly exclude the possibility that the examinee has been naturally infected with the virus used as vaccine.
We obtained very simple results. Only two types of Abs were isolated with no exceptions. The first type is the products of B cells newly induced through vaccination and the second type is the products of long-lived memory B cells established before vaccination. While there should be more clones that we have overlooked in this screening, it is unlikely that we have specifically overlooked clones with characteristics very different from those listed in Figure 2. For example, Abs that bind to HA of seasonal H1N1 virus but not to HA of pandemic virus were not isolated in the screening 4 (before vaccination, with seasonal virus), and all of the 16 clones were the second type of Abs. Since the library used in this screening was constructed from B cells before vaccination,  expansion of the first type of B cells through vaccination did not influence the characteristics of clones isolated from the screening 4. Thus, the number of B cells that produce Abs that bind to the globular head of HA of seasonal H1N1 virus should have been at a low level, even if they existed. This conclusion is further supported by the following observation. In the serum before vaccination, the  virus-neutralizing activity against seasonal H1N1 virus examined by focus reduction assay was at a detectable level, but the HI activity was not detected (Table 1). Since the surrounding regions of the sialic acid-binding pocket are immunogenically very potent, B cells producing Abs that bind to the epitopes could be preferentially generated. Therefore, these results should have reflected the selection mechanism of long-lived memory cells.
Our results also indicate that majority of the memory cells present in the donor's body produce Abs with similar characteristics, that is, they utilize the 1-69 V H gene, bind to the stem of HA, and neutralize both H1N1 and H5N1 viruses. They are major players in protection against H1N1 virus infection in the donor's body. We analyzed a person who had experienced influenza disease in their youth but never suffered from this disease afterwards. Since he has never been vaccinated against influenza, the Ab repertoire formed in his body was generated only by the effects of infection with live viruses. This observation is consistent with the following hypothesis. In our previous study [8], we analyzed the repertoire of neutralizing Abs against H3N2 viruses in a donor born in 1960. Many anti-HA Abs were isolated, and they were divided into three major groups with three distinct strain specificity: 1968-1973, 1977-1993 and 1997-2003. While five sites, such as A, B, C, D, and E, located on the globular head of HA have been identified as neutralizing epitopes [18,19], most of the clones that neutralize the 1977-1993 strains bound to site C [20]. Between 1977 and 1993, many mutations were introduced into sites A and B, but no mutations were introduced into site C. In order to explain these observations, we proposed the following hypothesis [20]. After a set of B cells producing Abs that can neutralize the viruses are generated by infection and/or vaccination, they will take various courses under further stimulation with the Ags. Some B cells disappear but others remain as memory cells. Furthermore, there should be long-lived memory cells and short-lived memory cells. Humans who experience an outbreak of flu almost every year have opportunities to be infected by novel influenza viruses that have drifted away from previous viruses. Some memory cells produce Abs that are able to neutralize the novel viruses, but others produce Abs that cannot neutralize them. They would be selected through the presence or absence of stimulation with the Ags. According to this hypothesis, the stability of the epitope would greatly affect the fate of memory B cells. As already shown by other groups [15,16], the epitope recognized by 1-69Abs is extremely stable among group 1 viruses. Thus, the cells producing 1-69Abs were selected as long-lived memory cells.
We analyzed only one person in this study. Therefore, the observations described in this paper should not be generalized at present. However, all humans should have potential for generating 1-69Abs that can broadly neutralize group 1 viruses, since only the 1-69 V H gene is required for producing a broadly neutralizing Ab without participation of the V L domain in forming the Ag-binding site [15,16], and furthermore, the requirements of the CDR3 sequence in V H for binding to the HA stem appeared to be limited [15,16]. Thus, we propose that the resistance of people against influenza infections is explained by acquisition of the ability to produce Abs that bind to the stable epitope and that many people show the resistance against influenza since humans are able to easily generate 1-69Abs that neutralize all of group 1 viruses. The present study gave the example only for resistance against group 1 viruses. If this is the case, humans should also have abilities to generate Abs that can neutralize all H3 viruses. However, there have been few papers reporting isolation of broadly neutralizing Abs against H3 viruses from humans. CR8020 reported by Ekiert et al. [21] showed broad neutralizing activity against group 2 viruses including H3, and it binds to the HA stem distinct from the epitope recognized by 1-69Abs. F16 reported by Corti et al. [22] neutralized both group 1 and group 2 viruses, and it binds to a conserved epitope in the F subdomain of HA. In our previous studies [8,14,20], we analyzed B lymphocytes collected from three donors born in 1944, 1960, and 1974 and revealed the repertoire of neutralizing Abs against H3 viruses. As summarized above, the Abs produced in two donors born in 1960 and 1944 were divided into three major groups with three distinct strain specificities: 1968-1973, 1977-1993 and 1997-2003. On the other hand, the Abs in the donor born in 1974 were composed of only four different types. Two of them showed narrow strain specificity: most strongly bound to the 1973 strain, and bound to the 1997 to 2003 strains. The other two types showed extremely broad strain specificity: bound to all of H3 strains, and bound strongly to all of H3 strains, moreover, weakly to group 1 viruses. These observations could be interpreted as follows: since he obtained B cells producing Abs that neutralize all of H3 viruses, the B cells producing Abs that specifically neutralize the viruses present between 1980 and 1995 disappeared.
How to select the B cells that produce Abs binding to the stable epitope as long-lived memory B cells would be explained by the following observations. HI activity against H1N1 pandemic virus started to appear in the serum 2 weeks after the vaccination. On the other hand, in the case of virus-neutralizing activity measured by focus reduction assay, the activity against H1N1 pandemic virus already started to increase 1 week after vaccination and reached a plateau 2 weeks after vaccination. When viruses infect a person, memory B cells that produce Abs with the ability to neutralize the infected viruses are stimulated to grow earlier than the birth of B cells newly induced by infection with the viruses.
While further experiments are required to examine whether Abs produced in the type 2 clones are strong enough for preventing infection with future pandemics caused by highly pathogenic avian influenza (HPAI) H5N1 virus, we may expect that the presence of type 2 clones as memory cells could be helpful for preventing expansion of the pandemic viruses in their bodies. Thus, we propose that the strategy for protection against the H5N1 pandemic should be designed according to the immunological profile of each individual. Presence of anti-HA Abs utilizing the 1-69 V H gene could be a useful indicator for presence of broadly neutralizing Abs and anti-idiotypic Abs against 1-69Abs could be the reagent.