Biol. Bull. 207: 172. (October 2004)
© 2004 Marine Biological Laboratory
Blood Clotting in Limulus Immunity: Physiological Impairment of Clot-Entrapped Bacteria
Peter A. Bosniak1 and
Peter B. Armstrong2
1 Hunter College, New York City, New York
2 University of California, Davis, California
The fibrillar extracellular blood clot is presumed to function in hemostasis, wound healing, and immunity. In that latter regard, the clot immobilizes microbes invading through wounds to prevent their systemic dispersal in the host. In the American horseshoe crab, Limulus polyphemus, the clot has been shown to immobilize bacteria so effectively that bacteria embedded in the clot lack even thermal (Brownian) motion. Two processes can be imagined to account for immobilization: (1) bacteria within the clot are physically entrapped by an array of clot fibrils so dense as to prevent any motion, or (2) the bacteria adhere strongly to the clot fibrils so as to prevent motion. The role of bacterial adherence was examined by determining the extent to which free-swimming bacteria attach to the surface of a pre-formed clot. Blood clots were produced by collecting blood onto coverglasses immersed in 0.5 M NaCl in a petri dish. Blood cells attach to the coverglass, degranulate, and produce the extracellular clot as a layer above the attached cells. The clot-bearing coverslips were then mounted on a microscope slide in a simple perfusion chamber that allowed exchange of the bathing buffer and microscopic examination with oil immersion objectives. The marine bacterium Vibrio alginolyticus, an opportunistic pathogen of Limulus, was grown in suspension culture and used in the log phase of culture growth. Attachment to the surface of the clot was studied by focusing at the blood clot/buffer interface with a 100x oil immersion phase contrast objective; bacteria were then introduced into the observation chamber. Vibrio were observed to swim freely until contacting the clot surface, at which instant a majority attached to the surface of the clot and were immobilized. Some attached bacteria maintained the use of flagellae, as evidenced by rapid spinning around an attachment point, while others displayed an absence of any motion, including a loss of Brownian motion. Bacteria attach to the native clot and to clots extracted with 0.5% Triton X-100. Attachment is resistant to calcium chelation, 2 M NaCl, and 8 M urea. These results indicate that adherence to the clot is sufficient to produce bacterial immobilization and that physical entrapment is not required.
This research was supported by NSF Grant 0344360 (PBA) and a Howard Hughes Undergraduate Fellowship (PAB).
