Antimicrobial defense mechanisms in the horseshoe crab,Limulus polyphemus: Effect of sodium chloride on bactericidal activity

Bactericidal activity of heat-derived (60°C) extracts of Limulus amoebocyte lysate (LAL) was found to be inhibited by low molar (10 to 100 mm) concentrations of the monovalent cations Na⁺ and K⁺. The protective effect of Na⁺ was shown to be species dependent and inversely proportional to the sensitivity of the test microorganism as determined by bactericidal titer. The effect of Na⁺ (100 mm) on bactericidal activity in whole (unheated) LAL, when assayed against a sensitive species (Escherichia coli), was negligible and in direct contrast to data obtained when using 60°C extracts of LAL. Increasing the concentration of Na⁺ to reflect levels in L. polyphemus blood (400 to 500 mm) afforded only minimal protection for sensitive bacterial species. These data suggested that whole LAL may contain a heat-labile substance (protein?) capable of binding Na⁺, obviating the inhibitory effect of Na⁺ on bactericidal activity. Further evidence for the existence of this cation-binding substance was obtained when 55°C extracts of LAL were found refractory to Na⁺ concentrations which totally abolished bactericidal activity in 60°C extracts. The mechanism of Na⁺ inhibition of bactericidal activity and the role of the cation-binding substance in L. polyphemus host defense against bacterial invasion and dissemination is discussed.