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

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Abstract
Sensitized cells of Vibrio cholerae , were killed and converted to spheroplasts by C[image] alone without the addition of lysozyme. Salmonella typhi Ty2 required 130 ug of the enzyme for conversion of 50 percent of its C[image] killed cells to spheroplasts, whereas only 2.3 ug were needed with comparable cells of S. typhi 0901. Also of significance was the marked difference in the slope of the dose-response relationship observed with S. typhi Ty2 in comparison with the other organisms tested. Possibly the low slope with strain Ty2 may be related to its high level of Vi antigen which results in the inaccessibility of its lysozyme substrate either as a result of physical or chemical forces. Other possible factors contributing to differences in lysozyme sensitivity of C[image] killed cells include quantitative as well as qualitative differences in the cell wall glycosamino-peptides that are the substrates of lysozyme. Although the immune bactericidal reaction and lysis of V. cholerae was indistinguishable experimentally, the killing of S. typhi organisms was attributed to C[image] and their subsequent lysis by lysozyme. This distinction may result from differences in the anatomical structures in S. typhi attacked by C[image] and by lysozyme. The cell membrane was implicated as the site of damage by C[image]. The C[image] attack upon a bacterial cell which results in loss of permeability control and its death conceivably results in the uptake of water, swelling of the cells, and consequent exposure of the cell''s lysozyme substrate. Lysozyme degrades the cell wall''s insoluble polymers which constitute its rigid layer. Subsequent attack by lysozyme results in a loss of the cell''s structural rigidity which does not occur as a result of C[image] action alone except perhaps in the case of Vibrio species. The cell wall of V. cholerae, unlike the cell walls of the Enterobacteriaceae, may require an intact cell membrane to maintain its rigidity. Although data for V. cholerae are not available, the percentage of lipid in the cell wall of Vibrio metchnikovi is 11.2 and the percentage of polysaccharide is 12.3, while comparable percentages for Salmonella gallinarum are 22 and 28 respectively. It is likely that the cell walls of Vibrio species were less rigid and more easily ruptured. Other possible alternatives include the activation of a cell wall autolytic process by C or the presence of lytic enzymes in serum, other than lysozyme, that are not removable by absorption with bentonite.