Loss of pigmentation in Shigella flexneri 2a is correlated with loss of virulence and virulence-associated plasmid

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Abstract
The relationship between the virulence of S. flexneri 2a and the ability of strains of S. flexneri 2a to absorb Congo red was examined. Spontaneous nonpigmented (i.e., unable to bind Congo red [Pcr-]) derivatives of a virulent, pigmented (Pcr+) strain of S. flexneri 2a were isolated and assayed for virulence as determined by their ability to invade epithelial cells. All Per- mutants examined lost the ability to invade epithelial cells and were thus avirulent. Agarose gel electrophoresis of plasmid DNA from these avirulent, Pcr- mutants showed that the majority of these strains had lost a plasmid band corresponding to a virulence-associated plasmid, pSf2a140. In many of the mutants, concomitant loss of pigmentation, virulence and pSf2a140 was accompanied by the appearance of a new plasmid, smaller than pSf2a140. These new plasmids may be deletion derivatives of pSf2a140; loss of pigmentation and virulence may be associated with deletions in pSf2a140. Pcr- mutants were transduced to Pcr+; transductants which suppressed the Pcr- phenotype were isolated. None of the Pcr+ transductants regained the ability to invade epithelial cells. Several suppressors of the Pcr- phenotype were identified as mutations in cell wall biosynthesis. Although pigmentation is usually associated with virulence, genetic determinants unrelated to virulence may also affect the ability of the cell to bind Congo red. Therefore, the ability of S. flexneri 2a to bind Congo red does not necessarily imply the ability to invade epithelial cells; loss of ability to bind Congo red is accompanied by loss of virulence.