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Many rough mutants selected from isogenic smooth virulent and avirulent strains of Shigella flexneri were examined for virulence, using tissue culture infection and Sereny tests. Many of the rough mutants isolated from a virulent smooth strain were capable of penetrating tissue culture cells but incapable of producing a positive Sereny test. In contrast, we could not obtain from smooth avirulent strains any rough mutants capable of penetrating HeLa cells. Chemical analysis of lipopolysaccharide of some representative rough strains showed several patterns of sugar composition with a range of from Ra to Re chemotypes. There was no correlation between HeLa cell invasiveness and chemotypes of lipopolysaccharides, thus indicating little significance of oligosaccharides of the rough core as well as O antigens in the ability of S. flexneri to penetrate HeLa cells. When these invasive rough strains were given O antigen genes from a smooth avirulent Shigella Hfr strain, most of the transconjugants that expressed O antigens regained the ability to evoke keratoconjunctivitis in guinea pigs. We also examined the chromosomal loci of HeLa cell invasion by transferring carbohydrate fermentation genes of Escherichia coli K-12 Hfr and found two chromosomal loci, the rha and lac-gal regions, which control the ability to penetrate HeLa cells. These results suggested that O antigens and ability to penetrate tissue culture cells are independent and prerequisite attributes of virulence in Shigella flexneri to evoke keratoconjunctivitis in guinea pigs.

infection and immunity

HeLa cell invasiveness and O antigen of Shigella flexneri as separate and prerequisite attributes of virulence to evoke keratoconjunctivitis in guinea pigs