New sensitive detection method for lectin hemagglutination using microscopy.

The blood group system AB0 is determined by the composition of terminal oligosaccharides on red blood cells. Thanks to this structural feature, these groups can be recognized by saccharide-recognizing compounds. Lectins are proteins that are able to reversibly bind saccharide structures. They generally occur as multimers and are known as hemagglutination agents. Hemagglutination is a process in which blood cells are cross-linked via multivalent molecules. Apart from lectins, hemagglutination can also be caused by antibodies or viruses. A hemagglutination assay is commonly used for the detection of multivalent molecules that recognize blood cells, in order to search for their sugar specificity. It is traditionally performed on a microtiter plate, where the lectin solution is serially diluted and the lowest concentration of lectin causing agglutination is detected. This experimental set-up is utilized further for testing lectin specificity via a hemagglutination inhibition assay. We have developed a new way of detecting hemagglutination using microscopy, which was tested on purified lectins as well as cell lysates. Hemagglutination was performed on a microscope slide directly and detected using a microscope. Comparison with the standard hemagglutination assay using microtiter plates revealed that microscopic approach is faster and more robust and allows fast determination of lectin activities immediately in bacterial cytosols.

Burkholderia cenocepacia lectin A binding to heptoses from the bacterial lipopolysaccharide.

Bacteria from the Burkholderia cepacia complex (Bcc) cause highly contagious pneumonia among cystic fibrosis (CF) patients. Among them, Burkholderia cenocepacia is one of the most dangerous in the Bcc and is the most frequent cause of morbidity and mortality in CF patients. Indeed, it is responsible of "cepacia syndrome", a deadly exacerbation of infection, that is the main cause of poor outcomes in lung transplantation. Burkholderia cenocepacia produces several soluble lectins with specificity for fucosylated and mannosylated glycoconjugates. These lectins are present on the bacterial cell surface and it has been proposed that they bind to lipopolysaccharide epitopes. In this work, we report on the interaction of one B. cenocepacia lectin, BC2L-A, with heptose and other manno configured sugar residues. Saturation transfer difference NMR spectroscopy studies of BC2L-A with different mono- and disaccharides demonstrated the requirement of manno configuration with the hydroxyl or glycol group at C6 for the binding process. The crystal structure of BC2L-A complexed with the methyl-heptoside confirmed the location of the carbohydrate ring in the binding site and elucidated the orientation of the glycol tail, in agreement with NMR data. Titration calorimetry performed on monosaccharides, heptose disaccharides and bacterial heptose-containing oligosaccharides and polysaccharides confirmed that bacterial cell wall contains carbohydrate epitopes that can bind to BC2L-A. Additionally, the specific binding of fluorescent BC2L-A lectin on B. cenocepacia bacterial surface was demonstrated by microscopy.