Interaction between Leptospiral Lipopolysaccharide and Toll-like Receptor 2 in Pig Fibroblast Cell Line, and Inhibitory Effect of Antibody against Leptospiral Lipopolysaccharide on Interaction

Leptospiral lipopolysaccharide (L-LPS) has shown potency in activating toll-like receptor 2 (TLR2) in pig fibroblasts (PEFs_NCC1), and causes the expression of proinflammatory cytokines. However, the stimulation by L-LPS was weak eliciting the function of TLR2 sufficiently in pig innate immunity responses during Leptospira infection. In this study, the immune response of pig embryonic fibroblast cell line (PEFs_SV40) was investigated and was found to be the high immune response, thus TLR2 is the predominate receptor of L-LPS in pig cells. Further, we found a strategy using the antibody against L-LPS, to prevent L-LPS interaction with TLR2 in pig cells which could impact on immune activation

Mannose-Binding Lectin Inhibits the Motility of Pathogenic <i>Salmonella</i> by Affecting the Driving Forces of Motility and the Chemotactic Response

<div><p>Mannose-binding lectin (MBL) is a key pattern recognition molecule in the lectin pathway of the complement system, an important component of innate immunity. MBL functions as an opsonin which enhances the sequential immune process such as phagocytosis. We here report an inhibitory effect of MBL on the motility of pathogenic bacteria, which occurs by affecting the energy source required for motility and the signaling pathway of chemotaxis. When <i>Salmonella</i> cells were treated with a physiological concentration of MBL, their motile fraction and free-swimming speed decreased. Rotation assays of a single flagellum showed that the flagellar rotation rate was significantly reduced by the addition of MBL. Measurements of the intracellular pH and membrane potential revealed that MBL affected a driving force for the <i>Salmonella</i> flagellum, the electrochemical potential difference of protons. We also found that MBL treatment increased the reversal frequency of <i>Salmonella</i> flagellar rotation, which interfered with the relative positive chemotaxis toward an attractive substrate. We thus propose that the motility inhibition effect of MBL may be secondarily involved in the attack against pathogens, potentially facilitating the primary role of MBL in the complement system.</p></div

Effect of MBL on single flagellar rotation in wild-type <i>Salmonella</i>.

<p>(a) Schematic representation of the tethered cell assay. Typical results obtained from independent cells in motility buffer in the absence (b) and presence (c) of MBL are shown. The positive and negative values indicate counterclockwise (CCW) and clockwise (CW) rotations, respectively. (d) Ratio of CW to CCW rotations. Values are the average of 25 flagellar motors. Vertical lines denote the standard deviation. (e) Average rotation rates of <i>Salmonella</i> flagella measured 30 min after the addition of MBL to the motility medium. The data are the average values of 21 cells obtained in three independent trials. Vertical lines denote the standard deviation, and asterisks indicate the results of statistical analysis (*<i>P</i> < 0.05).</p

Effect of MBL on the fluorescence intensity of DiBAC₄(3).

<p>An increase in fluorescent intensity corresponds to a decrease in membrane potential. Fluorescent intensities at 517 nm obtained by excitation at 495 nm were determined using a fluorescence plate reader. The values are relative to the fluorescent intensity obtained in the motility buffer without MBL (−MBL). The average values of triplicate experiments are shown. Vertical lines are the standard deviation. The Student <i>t</i>-test was performed to indicate a significant difference between the results of −MBL and +MBL (*<i>P</i> < 0.05).</p

Intracellular pH measured using EGFP.

<p>Absorbance of EGFP at 490 nm was determined under each condition. Relative values of absorbance compared to the results obtained in the motility buffer (pH 7.0) without MBL (−MBL) are shown. A decrease in the absorption indicates that the intracellular pH is lowered (see main text). The average values of triplicate experiments are shown. Vertical lines denote the standard deviation, and the Student <i>t</i>-test was performed to evaluate the significance of the difference from the result obtained in the absence of MBL (*<i>P</i> < 0.05, **<i>P</i> < 0.01).</p

Motility of <i>Salmonella</i> was affected by the addition of MBL.

<p>(a) Swimming fraction. More than 100 cells were analyzed 30 min after starting an experiment. The average values of three independent experiments are shown. The agglutination was observed under the microscope and evaluated as shown in parenthesis as follows: −, no agglutination; +, partially agglutinated but some cells displaying motility; ++, completely agglutinated (b) Time course of the swimming speed measured in the presence of 2.5 μg/ml MBL. The time displayed on the horizontal axis is one after the addition of MBL. Rectangles and triangles represent MBL-treated cells and the control group, respectively. The average values of more than 100 cells are shown. Vertical lines denote the standard deviation. Statistical analysis (the Student’s <i>t</i>-test) was performed to evaluate the significance of the difference from the data of the control at each concentration (*<i>P</i> < 0.05, **<i>P</i> < 0.01).</p

Chemotaxis assay of <i>Salmonella</i>.

<p>(a) Schematic presentation of the flow chamber used in the microscopic agar drop assay. (b) An attractive response of <i>Salmonella</i> to the agar drop containing 10 mM serine. Squares and triangles represent the results obtained in the presence and absence of MBL, respectively. The values represent the ratios of the number of cells near the agar drop at each time point relative to that at 0 min, immediately after starting the assay. Data are the average values of four independent experiments. Vertical lines are the standard deviation and the statistical analysis (the Student’s <i>t</i>-test) was preformed to evaluate the significance of the difference from the results of the control (*<i>P</i> < 0.05, **<i>P</i> < 0.01).</p

Effect of MBL on flagellar rotation in the chemotaxis-deficient mutant of <i>Salmonella</i>.

<p>All of the experimental conditions are the same as those described for the assay of the wild-type strain (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154165#pone.0154165.g002" target="_blank">Fig 2</a>). Typical results obtained from independent cells in motility buffer in the absence (a) and presence (b) of MBL are shown. (c) The average rotation rates. Vertical lines denote the standard deviation, and asterisks indicate the results of statistical analysis. Data represent the average value of 19 cells obtained in three independent trials (*<i>P</i> < 0.05).</p