IDENTIFICATION OF 2 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 dys entery. Two S. flexneri chromosomal loci that are required for these p rocesses were identified by screening a pool of TnphoA insertion mutan ts. These mutants were able to invade cultured epithelial cells but co uld not form mild-type plaques. Analysis of the nucleotide sequence in dicated that the sites of TnphoA insertion were within two different r egions 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, d ownstream of tonB. The mutant with the insertion at 70 min was named v psC because it showed an altered pattern of virulence protein secretio n. The vpsC mutant formed pinpoint-sized plaques, was defective in rec overy from infected tissue culture cells, and was sensitive to lysis b y the detergent sodium dodecyl sulfate. Recombinant plasmids carrying the S, flexneri vpsA, -B, and -C genes complemented all of the phenoty pes of the vpsC mutant. A mutation in vpsA resulted in the same phenot ype as the vpsC: mutation, suggesting that these two genes are part of a virulence operon in S. flexneri. The mutant with the insertion at 2 8 min was interrupted in the same open reading frame as S. flexneri is pA. This ispA mutant could not form plaques and was defective in bacte rial septation inside tissue culture cells.