Ability of smooth and rough variants of mycobacterium avium and M. intracellulare to multiply and survive intracellularly: role of C-mycosides

A spontaneous rough (Rg) mutant of M. avium ATCC 15769 was mutagenized using N-methyl-N-nitro-nitrosoguanidine (MNNG). Out of 54 clones initially chosen on the basis of their morphological appearance on the 7H11 agar, seven Rg clones were selected on the basis of their response to current biochemical tests, and were later characterized for their cell wall amphiphilic contents (analysis of loosely-bound surface lipids for mycosides, peptidolipids, phospholipids, and mycolic acids by thin layer chromatography, and fatty acids by gas chromatography), and ability to grow intracellularly inside J-774 macrophages. A parallel study was also performed on a previously reported Rg mutant of M. intracellulare serovar 20 (W. W. Barrow and P. J. Brennan, J. Bact. 150 (1982) 381–384) which lacked the ability to synthesize mycosides C (MYC⁻). The results obtained were compared to parental smooth (Sm) colony-types of the respective M. avium-intracellulare strains. Our results showed that neither the ninhydrin-reacting lipids (probably peptidolipids) nor the glycopeptidolipids (mycosides C) were primary factors responsible for the intracellular survival and multiplication of these bacteria. Ultrastructural studies showed that although the polysaccharide-rich outer wall layer (POL) in case of MYC⁻ Rg strain was not uniformly distributed and contained blebs, these bacteria formed the characteristic electron-transparent zone (ETZ) upon phagocytosis by macrophages. Furthermore, the multiplication of MYC⁻ Rg strain inside macrophages did not result in intracellular selection of MYC⁺ bacteria, nor in Rg to Sm transition.