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

Abstract

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 Tachypleus tridentatus 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 Escherichia coli 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 Pseudomonas aeruginosa and Klebsiella pneumoniae and Gram-positive Streptococcus pneumoniae 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 P. aeruginosa 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.

Inhibitory Effect of Multivalent Rhamnobiosides on Recombinant Horseshoe Crab Plasma Lectin Interactions with Pseudomonas aeruginosa PAO1

“…rHCPL has been previously reported to recognize bacteria P. aeruginosa PAO1;t his interaction can be inhibited by the presence of l-rhamnose but not other monosaccharides, which indicates that rHPL recognizes bacteria through the l-rhamnose moiety on the bacterial surface. [2] Here, 2mm synthetic multivalent rhamnobiosides with different rhamnose contents were used to inhibit binding between 0.5 mm rHPL and P. aeruginosa PAO1 and compared with l-rhamnose monosaccharide treatment ( Figure 2).…”

“…Inhibition of the binding between rHPL and P. aeruginosa PAO1 by various rhamnosides was assayed by using an ELISA, as described previously. [2] Briefly,as uspension of bacteria (5 10 7 cells well À1 )i nc oating buffer (0.1 m sodium carbonate/bicarbonate buffer,p H9.6) was added to flat-bottom 96-well microplates (Thermo Scientific, USA) and incubated at 4 8Co vernight. After blocking with 3% bovine serum albumin (BSA) in phosphatebuffered saline (PBS) that contained 0.05 %T ween 20 (PBST) at 37 8Cf or 2h,t he plates were washed four times with PBST.N ext, rHPL (25 mL, 1 mm)a nd at wofold indicated concentration of lrhamnose (25 mL; Sigma-Aldrich, USA) or synthetic rhamnosides were first incubated at 37 8Cf or 30 min, then the mixture was added to the bacteria in the wells and kept at 37 8Cf or af urther 1h.F inally,m onoclonal anti-His (1:5000;C lontech) in PBST was added and the cells were incubated at 37 8Cf or 1h,then horseradish-peroxidase-conjugated anti-mouse immunoglobulin (IgG) (1:5000;J ackson Lab) in PBST was added.…”

“…This rHPL is demonstrated to bind selectively to certain bacteria, such as Gram-negative bacterium Pseudomonas aeruginosa PAO1 and Gram-positive bacterium Listeria monocytogenes.I nterestingly,i ts bacterial recognition activities occur through specific molecular recognition of lrhamnose in pathogen-associated molecularp atterns (PAMPs) on the bacterial surface. [2] l-Rhamnosei sa6-deoxyhexose commonly found in the cell walls and capsules of many pathogenic bacteria. In addition, rhamnose is an importantu nit in PAMPs and plays crucial roles in fundamental aspects of bacterial physiology,such as antibiotic resistance.…”

“…[9][10][11] The assembly of rhamnoclusters from the structural elements wase nvisaged by two consecutive 1,3-dipolar azidealkyne cycloaddition click reactions. To obtain tri-and tetravalent derivativeso fvarious architectures,simple tri-and tetraols, such as methyl gallate, pentaerythritol, and N-Boc-protected tris(hydroxymethyl)aminomethane( Tris) equipped with propargyl moieties (2)(3)(4), were chosen as the multivalent platforms. Tris derivative 4 has the advantage of also having an additional functional group that, after deprotection,p rovides an opportunity for further functionalization of the multivalent derivatives.…”

Marine lectins and their medicinal applications

“…Although the mechanisms of the marine organism defense systems have been revealed for some lectins, the possibility to apply those to other antimicrobial mechanisms remains to be examined. Further studies on molecular mechanisms of action (Li et al 2015;Ng et al 2014), structure-function relationships (Huang et al 2015;Zhou and Sun 2015), and clinical trials can help researchers in investigating the therapeutic effects and toxicity of lectins. It is also worthwhile to check if lectins have synergism with existing antimicrobial agents.…”