Use of real-time, label-free analysis in revealing low-affinity binding to blood group antigens by Helicobacter pylori.

Infectious diseases are often initiated by microbial adherence that is mediated by the binding of attachment molecules, termed adhesins, to cell surface receptors on host cells. We present an experimental system, oblique-incidence reflectivity difference (OI-RD) microscopy, which allows the detection of novel, low-affinity microbial attachment mechanisms that may be essential for infectious processes. OI-RD microscopy was used to analyze direct binding of the oncopathogen, Helicobacter pylori ( H. pylori ) to immobilized glycoconjugates in real time with no need for labeling tags. The results suggest the presence of additional Lewis b blood group antigen (Le(b)) binding adhesins that have not been detected previously. OI-RD microscopy also confirmed the high-affinity binding of H. pylori outer-membrane protein BabA to Le(b). The OI-RD microscopy method is broadly applicable to real-time characterization of intact microbial binding to host receptors and offers new strategies to elucidate the molecular interactions of infectious agents with human host cells.

[1]  E. Kuipers,et al.  Pathogenesis of Helicobacter pylori Infection , 2006, Clinical Microbiology Reviews.

[2]  C. Prinz,et al.  Clinical relevance of the Helicobacter pylori gene for blood-group antigen-binding adhesin. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[3]  Kit S. Lam,et al.  Oblique-incidence reflectivity difference microscope for label-free high-throughput detection of biochemical reactions in a microarray format. , 2007 .

[4]  Y.S. Sun,et al.  Effect of fluorescently labeling protein probes on kinetics of protein-ligand reactions , 2008, 2008 Conference on Lasers and Electro-Optics and 2008 Conference on Quantum Electronics and Laser Science.

[5]  J. Parsonnet,et al.  Determination of the Infectious Dose ofHelicobacter pylori during Primary and Secondary Infection in Rhesus Monkeys (Macaca mulatta) , 2001, Infection and Immunity.

[6]  James C Paulson,et al.  Sweet spots in functional glycomics , 2006, Nature chemical biology.

[7]  C. Olsen,et al.  Role of ABO Secretor Status in Mucosal Innate Immunity and H. pylori Infection , 2008, PLoS pathogens.

[8]  K. Lam,et al.  A novel high-throughput scanning microscope for label-free detection of protein and small-molecule chemical microarrays. , 2008, The Review of scientific instruments.

[9]  R. Hancock,et al.  Comparative Genomics of Helicobacter pylori: Analysis of the Outer Membrane Protein Families , 2000, Infection and Immunity.

[10]  S. Normark,et al.  Attachment of Helicobacter pylori to human gastric epithelium mediated by blood group antigens. , 1993, Science.

[11]  J. Holmgren,et al.  Interaction of cholera toxin and membrane GM1 ganglioside of small intestine. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[12]  S. Normark,et al.  Helicobacter pylori: molecular basis for host recognition and bacterial adherence. , 1994, Trends in microbiology.

[13]  Kit S Lam,et al.  Screening small-molecule compound microarrays for protein ligands without fluorescence labeling with a high-throughput scanning microscope. , 2010, Journal of biomedical optics.

[14]  R. Schnaar,et al.  High Affinity Binding of the Entamoeba histolytica Lectin to Polyvalent N-Acetylgalactosaminides (*) , 1995, The Journal of Biological Chemistry.

[15]  M. Syvanen,et al.  Modification of Helicobacter pylori outer membrane protein expression during experimental infection of rhesus macaques , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[16]  M. Unemo,et al.  Helicobacter pylori adhesion to carbohydrates. , 2006, Methods in enzymology.

[17]  Chung-Yi Wu,et al.  Glycan arrays: biological and medical applications , 2008, Current Opinion in Chemical Biology.

[18]  B. Marshall,et al.  Expression of the BabA Adhesin during Experimental Infection with Helicobacter pylori , 2010, Infection and Immunity.

[19]  J P Landry,et al.  Protein reactions with surface-bound molecular targets detected by oblique-incidence reflectivity difference microscopes. , 2008, Applied optics.

[20]  L. Engstrand,et al.  Helicobacter pylori adhesin binding fucosylated histo-blood group antigens revealed by retagging. , 1998, Science.

[21]  S. Normark,et al.  Characterization of microbial host receptors. , 1994, Methods in enzymology.

[22]  M. Blaser,et al.  Functional Adaptation of BabA, the H. pylori ABO Blood Group Antigen Binding Adhesin , 2004, Science.

[23]  J P Landry,et al.  Macromolecular scaffolds for immobilizing small molecule microarrays in label-free detection of protein-ligand interactions on solid support. , 2009, Analytical chemistry.

[24]  L. Hammarström,et al.  Helicobacter pylori SabA Adhesin in Persistent Infection and Chronic Inflammation , 2002, Science.

[25]  J P Landry,et al.  Label-free detection of microarrays of biomolecules by oblique-incidence reflectivity difference microscopy. , 2004, Optics letters.