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Phagocytosis Stimulation by an Extracellular Product of Virulent Shigella flexneri 2a

It has been made clear that the cell infectivity of virulent Shigella flexneri 2a was stimulated by divalent cations such as Mg++ or Ca++ added to the growth medium (16, 17). The stimulative effect of the divalent cations was confirmed by the fact that addition of EDTA to the growth medium gave a reduction of the increased cell infectivity of the bacilli. Furthermore, the reductive effect of EDTA was noticed in the keratoconjunctival infection system in guinea pigs, i.e., the development and severity of lesions were suppressed by treatment with lotions of EDTA (17). These facts caused us to speculate that cell invasiveness of virulent Shigella might be mediated by a certain extracellular product specifically activated, stabilized, or mediated in its formation by Mg++ and/or Ca++. From this point of view, we examined the effect of crude extracellular products of a virulent strain of S. flexneri 2a on the cell infectivity of a few strains of Shigella. The bacteria used in this study were S. flexneri 2a, strain 5503, and Shigella sonnei, strains 74-306 and 74-487. Strain 5503 used mainly was isolated from the colonal specimens of a cynomolgus monkey which died of natural bacillary dysentery (8). It has been maintained in the lyophilized state as a standard strain in our laboratory. Strains 74-306 and 74-487 obtained from Dr. Zen-Yoji, The First Department of Bacteriology, Tokyo Metropolitan Research Laboratories of Public Health, were isolated from patients with sporadic bacillary dysentery in 1974 in Tokyo, and have also been maintained in the lyophilized state. Strain 5503 was subcultured in Luria broth (4) supplemented with MgSO4 at a final concentration of 5 mm at 37 C for 18 hr. One milliliter of the broth culture was then inoculated into 400 ml of the same broth, and incubated at 37 C for a further 18 hr with shaking. The shaking culture was centrifuged at 10,000 rpm for 15 min, and the supernatant fluid was sterilized by millipore filtration. The filtrate was then lyophilized after sufficient dialysis against distilled water and named as LB ( +). As controls, the organisms were cultivated in the broth without MgSO4, and the lyophilized sample named LB (-) was prepared by the same procedure as mentioned above. These