Comparison between motility (panel A) and track linearity (panel B) of ST maintained alone and ST in the presence of <i>S. b</i>-B or beads added during infection.

<p>Video-microscopic acquisitions were made 30 min PI, and 100 ST were tracked in each condition. In panel A are presented the statistical comparison between the average speeds of ST alone versus the average speeds of ST maintain in the presence of <i>S. b</i>-B or beads. In panel B are presented the quantification of different type of tracks: LT, CT or RT. The percentages marked in each column correspond to: the number of specific track type/the total number of track for each condition ×100. The data derived from a sequence using MTrack J processing software.</p

Characteristics of the motility of the mutated strain M913.

<p>In panel A and B are presented the swimming trajectories of strain M913 incubated alone with T84 cells (A) or cells incubated overnight (ON) with <i>S.b</i>-B and exposed to the strain M913 (B) derived from a sequence using MTrackJ. In panel C are presented the statistical comparisons between the average speed of strain M913 alone versus the average speed of strain M913 in the presence of <i>S.b</i>-B(ON). In panel D are presented the quantification of different type of trajectories of strain M913 alone versus the average speed of strain M913 in the presence of <i>S.b</i>-B(ON). Data of panels C and D derived from a sequence using MTrack J processing software. In panel E are presented the invasion of T84 cells infected one hour with the strain M913 alone or with the wild type strain SL 1344 alone. Invasion was assessed by the gentamicin protection method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033796#pone.0033796-Martins1" target="_blank">[23]</a>. % of invasion was normalized versus SL1344 as 100%. *statistically significant (P<0.05).</p

Swimming trajectories of ST incubated alone with T84 cells (A) or cells incubated overnight (ON) with <i>S.b</i>-B and exposed to ST (B) derived from a sequence using MTrack J processing software.

<p>Records were performed 60 min post infection (PI), and the time between consecutive images was 0.1 second. Using MTrackJ software we determined the locations for each bacterium, and this information was then translated into coordinates (x,y) for each bacterial cell and the process was repeated in times series. The 2D trajectories of each bacterial cell were represented; different colours represent different trajectories. Each trajectory has it own number. Arrows in panel B indicate “rotating” ST trajectories.</p

Estimation of the surfaces of <i>S.b</i>-B, ST and beads.

<p>Estimation of the surface of <i>S.b</i>-B, ST and beads was made by using ImageJ plugin (more details in supplemental data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033796#pone-0033796-g001" target="_blank">Figure 1</a>).</p

Modification of ST invasion by <i>S.b</i>-B, beads or <i>S.b</i>-B-CM.

<p>T84 cells were infected 60 min with the wild type strain SL 1344 alone or in the presence of <i>S.b</i>-B, beads or the conditioned medium by the yeast (<i>S.b</i>-B-CM) added during the infection. Experiments were also performed in cell incubated overnight (ON) with <i>S.b</i>–B (<i>S.b</i>-B(ON)) prior infection. At least beads and <i>S.b</i>-B-CM were added together during infection with ST. Invasion was assessed by the gentamicin protection method (23). % of invasion was calculated as intracellular bacteria/CFU of ST added by well (10⁷ CFU/well). * Indicates statistical difference <i>vs</i> ST-alone infected cells (P<0.05) (n = 4).</p>#<p>Indicates statistical difference <i>vs</i> beads+SL1344 infected cells or <i>S.b</i>-B-CM+SL1344 (P<0.05) (n = 3).</p

Strains used in this study.

<p>Strains used in this study.</p

Model of interaction between <i>S.b</i>-B and ST.

<p>The collision between “smooth” swimming ST and the yeast, induces a change in flagella organisation that modifies ST motility in a ‘tumble” mode, decreasing the speed of bacteria, changing the trajectories and finally enabling the adhesion of ST to yeast as previously observed by electronic microscopy <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033796#pone.0033796-Martins1" target="_blank">[23]</a>.</p

Determination of the linearity of bacterial trajectories.

<p>Determination of the linearity of bacterial trajectories.</p

Comparison between the track linearity (LT) of ST incubated alone with T84 (A) or cells incubated overnight (ON) with <i>S.b</i>-B (<i>S.b</i>-B(ON)) and exposed to ST (B).

<p>Tracks are presented in descending order implying that track number in “x” axis did not correspond to the track number attributed by Image-J. 187 ST- alone tracks are presented in panel A and 187 tracks of bacteria in the presence of <i>S. b</i>-B(ON) are presented in panel B. In panel C are represented the superposition of the all tracts from panel A and B. Quantification of different types of tracks of ST alone or ST in the presence of <i>S.b</i>-B are presented in panel D. The percentages marked in each column correspond to: the number of specific track type/the total number of tracks (187) for each condition ×100. The data derived from a sequence using MTrack J processing software. LT: linear trajectories; CT: curvilinear trajectories, RT: rotatory trajectories.</p

CNF1 activity confers curative immunoadjuvant properties.

<p>BALB/c mice were first infected with 3x10⁶ of stationary phase parasites. Fourteen days post-infection, mice were immunized via the nasal route with promastigote lysate (PL), wild-type CNF1 (WT CNF1), catalytically inactive CNF1 (mCNF1), PL plus either WT CNF1 (WT CNF1 + PL) or catalytically inactive CNF1 (mCNF1 + PL). The controls represent infected but non-immunized animals. Twenty-one and twenty-eight days post infection, the mice were immunized again. At day 42, the mice were sacrificed, and parasite numbers were determined by quantitative PCR using mouse spleen DNA extracts. The bars represent the mean cytokine production ± SEM. *: p<0.05, **: p<0,01, ***: p<0,001. n = 5.</p