Formation of Spheroplasts of Mycobacterium Tuberculosis by Lysozyme in Combination With Certain Enzymes of Rabbit Peritoneal Monocytes

Abstract

Spheroplasts of Mycobacterium tuberculosis were formed by culture of the organism in a nutritionally complete medium to which lysozyme and an extract obtained from rabbit peritoneal monocytes were added. The potentiating effect of the factor, or factors, present in the monocyte extract was lost upon prolonged storage at −20 °C or heat at 90 °C for 5 minutes, but was unaffected by dialysis. It was inactive in the presence of fluoride or oxalate. Enzymatic analysis of the monocyte extract showed the presence of lysozyme, acid phosphatase, phospholipase, and trypsin. When acid phosphatase or phospholipase were substituted for the monocyte extract in the lysozyme test system, spheroplast formation was obtained.

Mycobacterium

“…The accumulation ofenough host macromolecules about the surface of Phe I should render it somewhat indistinguishable from the host's self Most Phe I appear to have an intact cell wall. This is interesting in light of the fact that Willet and Thacore found that either acid phosphatase or phospholipase C could render H37Ra susceptible to lysozyme, with subsequent protoplast formation (1263) and, with high enough numbers of bacteria, even L-form development (1262). The addition of lysozyme to cell cultures of J-111 human monocytes has made possible the formation of protoplasts in these host cells (1147).…”

Protoplast formation of selected Mycobacterium smegmatis mutants by lysozyme in combination with methionine

“…The methods for removing the cell wall by lysozyme have been used with success on a variety of bacteria. However, protoplasts of mycobacterial cells were not readily prepared by lysozyme alone, since the complex nature of mycobacterial cell wall makes these organisms relatively resistant to the action of lysozyme (8,10). Some strains of rapidly growing mycobacteria and of strain BCG, when grown in the presence of lysozyme and glycine, become osmotically sensitive forms believed to be spheroplasts (1,(4)(5)(6).…”

Antigens of Mycobacterial Cell Walls

“…DISCUSSION Degradation with enzymes offers a way other than classical chemical extraction for the study of mycobacterial cell wall antigens. Although mycobacteria are relatively resistant to degradation by lysozyme, several investigators have reported alteration of isolated mycobacterial cell walls by this enzyme (8,14,16,17). In the work reported here, we wished clearly to distinguish between wall and nonwall antigens.…”