A detailed investigation of the amino acids of cell wall preparations obtained by various methods from the BCG strain is reported.1. "Non-peptidoglycan" amino acids represent about 1501, of the weight of crude, delipidated cell walls (Table 1) ; trypsin-chymotrypsin treated, delipidated cell walls contain mainly the peptidoglycan amino acid alanine, glutamic acid and meso-2,2'-diaminopimelic acid, the ratio Ala/A,pm being 1.5 and the ratio Glu/A,pm being 2.3, as compared with the expected values of 2 and 1 for a classical peptidoglycan; there are, however, equal molar amounts of D-glutamic acid and diaminopimelic acid and the excess of glutamic acid is the L-form.2. After enzymatic solubilization of the peptidoglycan by lysozyme or by Myxobacter AL, enzyme goo/, of the peptidoglycan amino acids are found in the soluble part ; this has been fractionated on Sephadex G-50 and in most of the fractions obtained the ratio Glu/A,pm is 1, as expect ed, but the ratio Ala/Agm is closer to 1 than to 2, indicating some other, not yet defined cross link to diaminopimelic acid.3. The insoluble residue obtained after lysozyme or Myxobacter AL, enzyme digestion contains a poly (L-glutamic acid) ; partial acid hydrolysis allows the solubilization of various L-glutamic acid oligopeptides, which have been studied by mass spectrometry, after N-acetylation and permethylation ; Fig. 3 shows a partial mass spectrum obtained from a peptide containing 6 or 7 glutamic acid residues, proving the sequence Glu-Glu-Glu-Glu ; pyroglutamic acid is observed in all spectra, indicating that at least the N-terminal glutamic acid residue is in a-linkage. The intact polymer could not be isolated, but the largest fragment obtained has an average chain length of eleven glutamic acid residues. 4. The presence of L-glutamic acid polymers seems to be restricted to human and bovine strains of Mycobacteria and to Mycobacterium kamaaii.
itThe basal structure of mycobacterial cell walls is made up of two covalently linked polymers: a peptidoglycan and a mycolate of an arabinogalactan [l-41. These polymers represent about 60°/, (w/w) of the weight of the cell wall. Besides this covalent structure, the main constituents of mycobacterial cell walls are free lipids (i.e. lipids which can be removed by neutral solvents) and amino acids which do not belong to the peptidoglycan. These amino acids which represent about 15O/, (w/w) of the weight of BCG cell walls seem to play an important role for their biological properties. Misaki [5] for instance has shown that, whereas crude BCG cells walls are capable of sensitizing guinea pigs to tuberculin and give a positive skin test in BCG-treated animals, these properties are lost after pronase treatment which removes most of the "non-peptidoglycan" amino acids. BCG [6,7] and crude cell walls of BCG [S] stimulate resistance to tumors and provoke tumor rejection, this latter property being perhaps related to the induction of a delayed hypersensitivity. We were thus prompted in connection with our investigations on the s...
