Poster Presentation BacPath 13: Molecular Analysis of Bacterial Pathogens Conference 2015

Neisseria gonorrhoeae and CR3: Probing the lectin functions of the I-domain of CD11b (#171)

Jessica Poole 1 , Chris Day 1 , Freda E.-C. Jen 1 , Lauren Hartley-Tassell 1 , Victor J. Torres 2 , Jennifer L. Edwards 3 , Michael P. Jennings 1
  1. Institute for Glycomics, Griffith University, Gold Coast, QLD, Australia
  2. Department of Microbiology, New York University School of Medicine, New York, NY, United States
  3. The Center for Microbial Pathogenesis, The Research Institute at Nationwide Children’s Hospital and The Department of Pediatrics, Ohio State University, Columbus, OH, United States

Neisseria gonorrhoeae is an exclusive human pathogen and the causative agent of the disease gonorrhea.  It has been previously shown that the type IV pili produced by the gonococcus has an important role in mediating adherence to human epithelial cells (1, 2, 3).  Pili of N. gonorrhoeae are post-translationally modified with a disaccharide, Gal(α1-3) 2,4-diacetamido-2,4,6-trideoxyhexose (3, 4).  This gonococcal pilin glycan participates in a protein-glycan interaction with the I-domain of complement receptor 3 (CR3, Mac-1, CD11b/CD18), which is a key receptor that mediates gonococcal adherence to primary human cervical epithelial (pex) cells (3).  Knowing the full lectin abilities of the I-domain would not only lead to a better understanding of the interplay between CR3 and the gonococcus, but other bacterial proteins that act through CR3.  A glycan binding profile of the I-domain was achieved using a recombinant human I-domain and glycan array analysis.  Surface plasmon resonance (SPR) was performed to verify the microarray results with a selection of glycans.  Array and SPR experiments were further verified by fluorescently labelling glycans and incubating on CHO cells over expressing CR3 and pex cells to determine if glycans bound CR3 ex vivo.  The recombinant human I-domain has found to have extensive lectin function as it bound over 100 structures on the array including structures contain α-linked galactose similar to the terminal glycosylation found on gonococcal pilin (terminal Galα1-3).    It was found that the recombinant human I-domain and the human CR3 complex bound to glycans with affinities ranging from 0.7 to 2.5µM and with off rates ranging from 0.01 to 1 x 10-9 1/s.  The affinities achieved from SPR matched the level of binding measured on the pex and CHO-CR3 cells.  These glycans, especially the high affinity interacting structures, could potentially be used to block gonococcal pilin glycan binding to CR3.

  1. Edwards, J. L., & Apicella, M. A. (2005). I-domain-containing integrins serve as pilus receptors for Neisseria gonorrhoeae adherence to human epithelial cells. Cell Microbiol, 7(8), 1197-1211.
  2. Edwards, J. L., Brown, E. J., Uk-Nham, S., Cannon, J. G., Blake, M. S., & Apicella, M. A. (2002). A co-operative interaction between Neisseria gonorrhoeae and complement receptor 3 mediates infection of primary cervical epithelial cells. Cell Microbiol, 4(9), 571-584.
  3. Jennings, M. P., Jen, F. E., Roddam, L. F., Apicella, M. A., & Edwards, J. L. (2011). Neisseria gonorrhoeae pilin glycan contributes to CR3 activation during challenge of primary cervical epithelial cells. Cell Microbiol, 13(6), 885-896.
  4. Hegge, F. T., Hitchen, P. G., Aas, F. E., Kristiansen, H., Lovold, C., Egge-Jacobsen, W., Koomey, M. (2004). Unique modifications with phosphocholine and phosphoethanolamine define alternate antigenic forms of Neisseria gonorrhoeae type IV pili. Proc Natl Acad Sci USA, 101(29), 10798-10803.