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1 Marine Biological Laboratory, Woods Hole, MA
2 Hunter College of the City University of New York, New York, NY
3 University of California, Davis, CA
* Corresponding author: pbarmstrong{at}ucdavis.edu
The fibrillar blood clot is an extracellular matrix established at sites of damage to the walls of the blood-vascular system. In humans, the clot is a polymer of the protein, fibrin. In the horseshoe crab, Limulus polyphemus, the clot is a meshwork of fibrillar polymers of the protein, coagulin (1). The blood clot functions to seal the wound to staunch bleeding, operates as a transient extracellular matrix for the migration of wound-healing epithelial and connective tissue cells, and serves as a barrier to entry of microbes into the interior of the animal via the wound. This study concerns the characterization of this last function for the coagulin blood clot of Limulus. We investigated the extent of immobilization of bacteria by the coagulin blood clot and the viability of the clot-entrapped bacteria.
Plasma was collected from adult horseshoe crabs under sterile, lipopolysaccharide-free conditions by cardiac puncture. Blood cells were removed immediately after collection and the plasma was sterile-filtered through a filter with a pore size of 0.22 µm (Corning, Inc., Cat # 430769). The marine bacterium Vibrio alginolyticus was grown in liquid culture on Marine Broth 2216 (Difco). Log-phase growing populations were established by culture for 12 h at room temperature. Blood clots were established by plating the cells contained in 1 drop of Limulus blood collected by cardiac puncture under sterile conditions in 2 ml of sterile 3% NaCl (Baxter Healthcare Corp., Deerfield, IL) in a 35-mm plastic petri dish (Falcon, Cat # 35-1008). Bacteria suspended in 0.1 M sucrose, 3% NaCl, equivalent to the bacteria contained in 250 µl of the original culture medium, were introduced into the culture dish before the blood cells were added to facilitate direct presentation of bacteria to the blood cells while the latter were attaching to the culture surface of the dish. Under these conditions, the blood cells degranulate to release coagulogen and the proteases that process it into coagulin, the form that polymerizes into the fibrillar clot (2). The result is the entrapment of numbers of bacterial cells in the meshwork of fibers of the coagulin clot. The fate of the entrapped bacteria was investigated by direct light microscopic observation. The two parameters of greatest interest were the immobilization of bacteria by the clot and the killing of clot-entrapped bacteria by components of the plasma operating in synergy with the clot.
In suspension, V. alginolyticus shows rapid flagellar-driven swimming locomotion. Bacteria entrapped in the coagulin clot are immotile, and are held so tightly as to lack even thermal (Brownian) motion. When bacteria were killed to eliminate swimming motility, more than 90% of the killed bacteria in suspension showed thermal (Brownian) motion; in contrast, all bacteria enmeshed in the clot were absolutely stationary, without thermal motion.
Entrapped bacteria survive and proliferate in clots maintained in artificial seawater, which is isotonic to Limulus blood. In a typical trial, 87% of the bacterial clusters enmeshed in the clot contained only one cell immediately after capture, but 54% of the bacterial clusters contained two or more cells by 4 h of incubation. Proliferation is rapid if the clot, with its cargo of entrapped bacteria, is transferred to bacterial culture medium (Fig. 1A, B). After 4 h of incubation of the clot with entrapped bacteria in culture medium, 84% of bacterial clusters contained two or more cells. However, bacteria were killed when the preparation of clot with entrapped bacteria was transferred to sterile Limulus plasma. Killing was monitored by loss of refractility of the bacterial cells under phase-contrast microscope examination (Plan 100/1.3 NA objective) (Fig. 1C, D). The non-refractile cells ("ghost cells") are presumed to be dead or seriously damaged. Loss of bacterial refractility shows a lag period of 1.5 h, after which killing is rapid. In a typical trial, cells appeared normal and strongly refractile at 1.5 h, but about 80% of the cells had become transparent at 2 h. We did not observe bacterial proliferation in the plasma-treated clot even after 8 h of incubation; whereas, as noted above, the seawater-treated clot did support bacterial growth. The LD50 was at a 25% dilution of plasma. Interestingly, plasma-based cytotoxicity was dependent on entrapment in the coagulin clot because free bacterial cells diluted into plasma remained phase-dense and presumably alive. These cells did lose the capacity for flagellar-generated motility, but showed the same capacity for proliferation when transferred to nutrient agar as did cells treated under equivalent conditions in plain seawater.
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In summary, we have defined two important contributions of the coagulin blood clot to immunity in Limulus. The clot immobilizes microbes, which presumably impedes their dissemination throughout the animal after gaining access via a wound. Plasma also shows an immobilizing action by its inhibition of flagellar swimming motility. Neither clot nor plasma alone kill the bacteria but the two synergize to effect the cytotoxic destruction of clot-entrapped microbes.
This research was supported by a grant MCB 26771 from the National Science Foundation (PBA) and a fellowship from the Howard Hughes Medical Institute Undergraduate Science Education Program in Biology, grant 52002679 (VI). We thank Ms. Alice Child, Mr. Joseph Lee, and Drs. William Cohen and Norman Wainwright for help with the research.
Literature Cited
This article has been cited by other articles:
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  Y. Matsuda, T. Koshiba, T. Osaki, H. Suyama, F. Arisaka, Y. Toh, and S.-i. Kawabata An Arthropod Cuticular Chitin-binding Protein Endows Injured Sites with Transglutaminase-dependent Mesh J. Biol. Chem., December 28, 2007; 282(52): 37316 - 37324. [Abstract] [Full Text] [PDF]
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Y. Matsuda, T. Osaki, T. Hashii, T. Koshiba, and S.-i. Kawabata A Cysteine-rich Protein from an Arthropod Stabilizes Clotting Mesh and Immobilizes Bacteria at Injury Sites J. Biol. Chem., November 16, 2007; 282(46): 33545 - 33552. [Abstract] [Full Text] [PDF]
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 P. B. Armstrong and M. T. Armstrong The Decorated Clot: Binding of Agents of the Innate Immune System to the Fibrils of the Limulus Blood Clot Biol. Bull., October 1, 2003; 205(2): 201 - 203. [Full Text] [PDF]
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