Binding activity of rHPL to laboratory-derived bacteria.

<p>(A) Gram-negative and (B) Gram-positive bacterial cells were seeded at 5×10⁷ cells/well, and 1 µM of rHPL was applied to the microplate wells. Subsequently, monoclonal anti-His (1∶5000) was used to detect rHPL bound to bacterial cells. Blank refers to wells containing buffer instead of rHPL. The values are the mean ± SD from triplicate experiments. ***<i>P</i><0.001 <i>versus</i> the corresponding blank data.</p

Parameter of inhibitory effect on L-Rhamnose and L-Rhamnose-BSA conjugate to rHPL- LPS/bacteria interaction.

<p>The values are the mean ± SD (%) from triplicate experiments.</p><p>*<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001 <i>versus</i> the rHPL only group (positive control).</p><p>Parameter of inhibitory effect on L-Rhamnose and L-Rhamnose-BSA conjugate to rHPL- LPS/bacteria interaction.</p

A Recombinant Horseshoe Crab Plasma Lectin Recognizes Specific Pathogen-Associated Molecular Patterns of Bacteria through Rhamnose

<div><p>Horseshoe crab is an ancient marine arthropod that, in the absence of a vertebrate-like immune system, relies solely on innate immune responses by defense molecules found in hemolymph plasma and granular hemocytes for host defense. A plasma lectin isolated from the hemolymph of Taiwanese <i>Tachypleus tridentatus</i> recognizes bacteria and lipopolysaccharides (LPSs), yet its structure and mechanism of action remain unclear, largely because of limited availability of horseshoe crabs and the lack of a heterogeneous expression system. In this study, we have successfully expressed and purified a soluble and functional recombinant horseshoe crab plasma lectin (rHPL) in an <i>Escherichia coli</i> system. Interestingly, rHPL bound not only to bacteria and LPSs like the native HPL but also to selective medically important pathogens isolated from clinical specimens, such as Gram-negative <i>Pseudomonas aeruginosa</i> and <i>Klebsiella pneumoniae</i> and Gram-positive <i>Streptococcus pneumoniae</i> serotypes. The binding was demonstrated to occur through a specific molecular interaction with rhamnose in pathogen-associated molecular patterns (PAMPs) on the bacterial surface. Additionally, rHPL inhibited the growth of <i>P. aeruginosa</i> PAO1 in a concentration-dependent manner. The results suggest that a specific protein-glycan interaction between rHPL and rhamnosyl residue may further facilitate development of novel diagnostic and therapeutic strategies for microbial pathogens.</p></div

LPS binding activity of rHPL expressed in <i>E. coli</i>.

<p>A total of 0.5 µg of each LPS was coated on microplate wells and detected with 1 µM rHPL (A) or 1 µM DTT-treated rHPL (B). Monoclonal anti-His (1∶5000) was used to detect the rHPL bound to LPSs. Blank refers to wells containing buffer instead of rHPL. The values are the mean ± SD from triplicate experiments. ***<i>P</i><0.001 <i>versus</i> the corresponding blank data.</p

Binding activity of rHPL to clinically-isolated bacteria.

<p>(A) Gram-negative and (B) Gram-positive bacterial cells were seeded and rHPL binding was analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115296#pone-0115296-g003" target="_blank">Fig. 3</a>. The values are the mean ± SD from triplicate experiments. Individual sample numbers are indicated. <i>P. aeruginosa</i> sero 10 LPS was used as a 100%-binding positive control and <i>E. coli</i> Top10F′ was used as negative control. Relative binding percentages are relative to the positive control. *<i>P</i><0.05 and ***<i>P</i><0.001 <i>versus</i> the corresponding blank data.</p

Inhibitory effect of L-Rhamnose monosaccharide and Rha-BSA on rHPL-LPS/bacteria interaction.

<p>A total of 0.5 µg of <i>E. coli</i> O55:B5 LPS (A), <i>E. coli</i> O26:B6 (B), <i>S.</i> typhimurium (C), <i>P. aeruginosa</i> (D), or 5×10⁷ cells <i>P. aeruginosa</i> PAO1 (E) was coated on 96-well microplates and incubated at 37°C for 3 h or at 4°C overnight. The microplates with the immobilized bacteria were washed, and unbound regions were blocked with BSA. Various concentrations of glycans or glycan-protein conjugates were incubated with 1 µM rHPL and then added to microplates. Anti-His (1∶5000) was used to detect rHPL binding to bacterial cells. Blank refers to microplate wells containing only buffer. The values are the mean ± SD from triplicate experiments. *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001 <i>versus</i> the rHPL only group (positive control).</p

Antibacterial activity of rHPL.

<p>Samples of <i>P. aeruginosa</i> and <i>S. aureus</i><b> were</b> mixed with rHPL at a final concentration of 0 µM (buffer only), 0.47 µM, 0.94 µM, 1.88 µM, 3.75 µM, 7.5 µM, and 15 µM and incubated at 37°C for 4 h. The antibacterial activity of rHPL was analyzed by plating serial dilutions of incubation mixture, and the CFUs were counted the following day. Control plate (0 µM) was defined as 100%-viable cells. Cell mortality was calculated as the decrease in the colony number compared with the control plate. The values are the mean ± SD from triplicate experiments.</p

Purification and characterization of rHPL expressed in <i>E. coli</i>.

<p>(A) After induction with 0.1 mM isopropyl β-D-1-thiogalactopyranoside at 16°C for 16 h, the supernatant containing rHPL was collected by centrifugation and subjected to purification by nickel-column chromatography. Aliquots of each fraction were analyzed by 15% (w/v) SDS-PAGE. The expected molecular weight of rHPL was 19.4 kDa. Lane M: molecular weight marker; Lane N: non-induction; Lane I: induction; Lane P: insoluble pellet; Lane S: supernatant; Lane F: binding flow-through; Lane W1: washing fraction 1; Lane W2: washing fraction 2; Lane E: eluent; Lane C: concentrated fraction. (B) Mass determination of rHPL was performed by MALDI-TOF MS in the electrospray ionization mode. rHPL (100 pmol) was acidified with 0.1% (v/v) formic acid in 50% acetonitrile, and the data were acquired over the mass-to-charge ratio (<i>m</i>/<i>z</i>) range of 0–26,000 under normal scan resolution (<i>x</i> axis), the relative intensity (a.u., arbitrary units) are shown on the <i>y</i> axis. The data from each spectra were summed and deconvoluted. (C) Secondary structure of rHPL was measured by Far-UV CD spectrum (260 nm–190 nm) with protein concentration of 25 µM at 16°C.</p

Glycan binding activity of rHPL expressed in E. coli.

<p>(A) Glycan microarray analyses were conducted by the Consortium for Functional Glycomics. rHPL at a concentration of 200 µg/ml was used in the analysis, and anti-His (1∶1000) was used as the primary antibody. The values are the mean ± SD from triplicate experiments. (B) Direct binding between L-Rha monohydrate and rHPL was verified by magnetic reduction (MR) assay. rHPL was conjugated on magnetic nanoparticles (MNPs) and the increase of MR signal with the titration of L-Rha concentrations ranging from 0.01 to 1000 ng/ml was measured. LPS of <i>P. aeruginosa</i> and D-Galactose were used as positive and negative control respectively.</p

Chemical structures of PAMPs of bacterial pathogens.

<p>(A) LPS of <i>K. oxytoca</i> strain TMN3. (B) LPS A band of <i>P. aeruginosa</i>. (C) Capsule of <i>S. pneumoniae</i> serotype 19A. (D) Capsule of <i>S. pneumoniae</i> serotype 19B. (E) Capsule of <i>S. pneumoniae</i> serotype 19F. (F) Capsule of <i>S. pneumoniae</i> serotype 23F. (G) LTA of <i>L. monocytogenes</i> ATCC 7644.</p