FIGURE 1.

Identification of Mycobacterium marinum genes for virulence using Dictyostelium. (A) Screening method of M. marinum NTUH-M6094 mutant library by Dictyostelium phagocytosis plaque assay. In six-well tissue culture plates containing SM agar, mid-log phase (OD₆₀₀ = 0.8–1.2) cultured M. marinum and fresh overnight-cultured Klebsiella aerogenes (diluted 10⁵-fold) were mixed in normal saline and then air-dried. Four hundred Dictyostelium cells were then added on the top of the bacterial lawn. The plates were incubated at 20°C for 6–8 days until phagocytosis plaques became visible. (B) Dictyostelium phagocytosis plaques on the M. marinum M6094 wild type two transposon mutants and M. smegmatis. In presence of wild-type M. marinum M6094, Dictyostelium failed to form phagocytosis plaques. In contrast, a clear phagocytosis plaque was observed on bacterial lawn with M. smegmatis (avirulence mycobacteria). Two mutants 2-C10 and 4-B11 permit the formation of a clear phagocytosis plaque on the bacterial lawn.