Figure 4

Human blood plasma coagulates on surface clusters of many Bacillus species, including B. anthracis, but not on control species of E. coli and S. aureus. (a) Chart quantifying the clot times of human blood plasma on clusters of bacteria in a microfluidic chamber in the absence of flow. (b,c) B. anthracis strains that do not produce secreted zinc metalloproteases NprB (ΔnprB) or InhA1 (ΔinhA1) have reduced ability to activate purified human prothrombin (b) and purified human factor X (c). (d) Purified prothrombin is activated by InhA1 purified from B. anthracis. (e) A graph quantifying the clot times of human blood plasma on Ames 35, ΔnprB and ΔinhA1 strains of B. anthracis shows that the Ames 35 control strain rapidly initiated coagulation. The ΔnprB strain also initiated coagulation, whereas the ΔinhA1 strain did not accelerate coagulation relative to background clotting (P < 0.001 for Ames 35 versus ΔinhA1). (f) A graph quantifying the clot times of human blood plasma on control Ames 35 and a ΔluxS strain shows that the ΔluxS strain rapidly initiated coagulation despite its inability to secrete autoinducer-2 (1), a quorum-sensing signaling molecule. Each data point (a,e,f) represents the clot time on a single large (>300 μm) cluster of bacteria in a microfluidic chamber that was measured by fluorescence microscopy. Clotting on some surface clusters occurred before the first images obtained at 3 min (a) or 2 min (e and f), as indicated by breaks in the y axis.