Isolation and Properties of a Macromolecular, Water-Soluble, Immuno-Adjuvant Fraction from the Cell Wall of Mycobacterium smegmatis

Abstract

Trypsin-and chymotrypsin-treated delipidated cell walls of Mycobacterium smegmatis were digested overnight with lysozyme. The water-soluble products thus obtained were filtered on a column of Sephadex G-50; the first peak, excluded from the column, has immunological adjuvant activity. The material of the excluded peak is obtained after lyophilization as a snowwhite, fluffy material, soluble in water and insoluble in organic solvents. It behaves as a slightly polydisperse macromolecule in an ultracentrifuge, with an approximate molecular weight of 20,000. All the constituents of this material are typical bacterial cell-wall constituents; thus, the water-soluble adjuvant is considered to be an "oligomer" of the cell wall.When added to Freund's incomplete adjuvant with an antigen (e.g., ovalbumin) and injected into hind-foot pads of guinea pigs, this water-soluble adjuvant increases the amount of precipitating antibodies and induces hypersensitivity to ovalbumin and the biosynthesis of -y2-type precipitating antibodies. The water-soluble material has a stronger adjuvant activity than equal amounts of whole bacteria, cell walls, or wax D, and seems to be the first well-defined, water-soluble, adjuvant-active fraction isolated from Mycobacteria. It is known, since the work of Coulaud (1) and Freund (2, 3), that the injection of killed mycobacteria in mineral oil emulsified with water mixed with an antigen increases the production of circulating antibodies against the same antigen, and induces a delayed hypersensitivity to that antigen (2, 4). The water-in-oil emulsion containing killed mycobacteria is currently used for animal experiments and is called Freund's complete adjuvant.Various attempts have been made to isolate the adjuvantactive component(s) of mycobacterial cells.Wax D, a chloroform-soluble fraction of the free lipids of human strains of M. tuberculosis, can replace mycobacteria in Freund's adjuvant (5, 6); these active wax D fractions contain a peptidoglycan linked to a mycolate of an arabinogalactan (7-10) *. Their structure is thus very similar to that of the mycobacterial cell wall (7, 8, for a review, see ref. 9); they are, in fact, considered to be produced by autolysis of the cell wall (7,9,10 In a typical experiment, 1 g of cell walls is suspended in 100 ml of 0.05 M ammonium acetate (pH 6.3) and is incubated overnight at 370 with 20 mg of lysozyme, in the presence of a few drops of toluene. The solubilized products are separated from the cell walls by filtration on a sintered-glass filter. The cell walls are incubated again overnight under the same conditions. About 10% of the total hexosamines are solubilized by these lysozyme treatments. The water-soluble products are then lyophilized several times to remove the buffer and are filtered on a column of Sephadex G-50 (2.5 X 80 cm) in 0.1 N acetic acid (Fig. 1). The first peak, excluded from the column, is WSA$. A second peak, emerging later, contains lower molecular weight substances and also has adjuvant activity, but it has not been s...

TLR, NLR Agonists, and Other Immune Modulators as Infectious Disease Vaccine Adjuvants

“…NLRs are a family of cytoplasmic PRRs that contain 3 distinct domains: C-terminal LRRs, Muramyl dipeptide (MDP), the minimal unit of peptidoglycan was first recognised as the minimum effective component of complete freund's adjuvant (CFA) in 1972 [40].…”

Adjuvant Activity of Mycobacterial Fractions

“…Recently, some investigators reported the isolation and purification of WSA of BCG and its biological properties [1,2,27,36]. WSA isolated by digestion with lysozyme has potent adjuvanticity in vivo, even though it lacks the mycolic acid moiety of CWS [1].…”

The Amino Acids of the Cell Wall of Mycobacterium tuberculosis var. bovis, Strain BCG

“…of the Peptidoglycan Bacteriolytic enzymes solubilize only part of the peptidoglycan of delipidated cell walls [22]. However, if these cell walls are submitted to a mild acid hydrolysis with 0.1 N HCl more than 90°/, of the peptidoglycan can be solubilized enzymatically [21,23].…”