Identification of Two Shigella flexneri Chromosomal Loci Involved in Intercellular Spreading

The ability of Shigella flexneri to multiply within colonic epithelial cells and spread to adjacent cells is essential for production of dysentery. Two S. flexneri chromosomal loci that are required for these processes were identified by screening a pool of TnphoA insertion mutants. These mutants were able to invade cultured epithelial cells but could not form wild-type plaques. Analysis of the nucleotide sequence indicated that the sites of TnphoA insertion were within two different regions that are almost identical to Escherichia coli K-12 chromosomal sequences of unknown functions. One region is located at 70 min on the E. coli chromosome, upstream of murZ, while the other is at 28 min, downstream of tonB. The mutant with the insertion at 70 min was named vpsC because it showed an altered pattern of virulence protein secretion. The vpsC mutant formed pinpoint-sized plaques, was defective in recovery from infected tissue culture cells, and was sensitive to lysis by the detergent sodium dodecyl sulfate. Recombinant plasmids carrying the S. flexneri vpsA, -B, and -C genes complemented all of the phenotypes of the vpsC mutant. A mutation in vpsA resulted in the same phenotype as the vpsC mutation, suggesting that these two genes are part of a virulence operon in S. flexneri. The mutant with the insertion at 28 min was interrupted in the same open reading frame as S. flexneri ispA. This ispA mutant could not form plaques and was defective in bacterial septation inside tissue culture cells.