Quantitative Studies on Spheroplast Formation by the Complement System and Lysozyme on Gram Negative Bacteria. ∗

Abstract

Gram negative bacteria of the genera Vibrio, Salmonella, Shigella, Escherichia, Brucella and Hemophilus may be killed by the complement (C′) system(l). Some of the members of these groups, particularly rough forms, are extremely sensitive to small amounts of fresh normal serum, which serves as a source of both natural antibody and C′. Other organisms are not killed unless antiserum is present in addition to C′, and a third group, under the usual experimental conditions, is refractory to C′ despite sensitization by anti-serum (2), Among the Enterobacteriaceae, resistance to normal serum has been associated with the capsular (K) antigenic content of the organism(3) and, more significantly, from a biomedical point of view, with the virulence of the organism (4).

Although the bactericidal and bacteriolytic reactions mediated by serum substances have not generally been distinguished, recent observations have indicated that the bactericidal reaction requires antibody and C, whereas the lysis of the killed cells also requires the enzyme lysozyme(5,6). C′activity is not unique in rendering cells of gram negative bacteria susceptible to lysozyme attack since starved cells at an abnormal pH(7) or treatment with polymyxin B sulfate(8) will accomplish this result. Obviously different mechanisms may lead to cell lysis by lysozyme.

Bacteria rendered non-viable by the C′system are not grossly distorted, but are lysed only upon subsequent addition of lysozyme, or in a stabilizing milieu, converted to spheroplasts (5). The quantitative relationships between the susceptibility of an organism to C′and to lysozyme have not been determined. It was not known whether cells killed by C′derived from relatively sensitive organisms were equally or differently sensitive to lysozyme. Consequently, cells of organisms of varying sensitivity to C′were subjected to its action and the amount of lysozyme required for conversion of those cells to sphero-plasts was determined(9).