Mutants of Shigellaflexneri defective in aerobactin-mediated iron transport were assayed for virulence in several model systems. A TnS insertion mutant was invasive in HeLa cells, lethal in the chicken embryo, and produced keratoconjunctivitis in the guinea pig, indicating little or no loss of ability to invade and multiply intracellularly. Although the mutant failed to grow in low-iron medium in vitro, growth equivalent to that of the wild type was observed in HeLa cell lysates. Thus, there appears to be sufficient available iron inside the HeLa cell to allow growth in the absence of siderophore synthesis. Possible host iron sources were tested, and both the mutant and wild type utilized hemin or hematin as a sole source of iron. Only the wild-type, aerobactin-producing strain could remove iron from transferrin or lactoferrin. Two deletion mutants were also assayed for virulence and were found to be avirulent for the chicken embryo. These deletions encompass flanking sequences as well as the aerobactin genes; therefore, adjacent genes may be required for virulence.

The iron uptake systems of Plesiomonas shigelloides strains were determined. Siderophore production was not detected by chemical or biological assays, and the strains tested were unable to use enterobactin, aerobactin, or vibriobactin for growth in low-iron media. Both hemin and hemoglobin supported full growth of the bacteria in media lacking other iron sources, but neither transferrin nor lactoferrin served as a source of iron. Hemolysin was detected, and the production of hemolysin was iron repressible. DNA sequences encoding hemolysin production and DNA sequences encoding the ability to use heme or hemoglobin as a sole source of iron were cloned from P. shigelloides and expressed in Escherichia coli. The abilities to use heme and hemoglobin as iron sources were closely linked, and the cloned sequences encoded the ability to transport the porphyrin, as well as iron, into the cells.

Wild-type isolates of Shigella flexneri bind the dye Congo red from solid media, thus producing red (Crb+) colonies. Mutants which fail to bind the dye produce white colonies (Crb-) and are avirulent in a variety of systems. In S. flexneri the ability to bind Congo red correlates with the ability to bind hemin and protoporphyrin IX. Binding of hemin by Crb+ S. flexneri was observed both in solid media and in liquid assays. Results of competition experiments suggest that Congo red and hemin bind to the same site on the bacterial cell and are retained on the cell surface. Binding of hemin by Crb+ S. flexneri is independent of hemin transport since both Crb+ and Crb- cells can utilize hemin as a sole source of iron. Both Crb- and Crb- organisms were able to grow in HeLa cell lysates, indicating that the gene(s) that is responsible for Congo red binding does not play a role in the acquisition of intracellular heme iron. By using the HeLa cell invasion system, the effect of hemin prebinding on the invasiveness of Crb+ S. flexneri was determined. Crb+ cells which had prebound hemin exhibited increased invasiveness, indicating a possible role for the crb gene product in the initial stages of invasion by S. flexneri.

The ability of Shigellaflexneri to bind Congo red or hemin is associated with virulence. A 101-kilodalton (kDa) protein responsible for this phenotype (Crb+) in S. flexneri was identified by a tetramethylbenzidine staining procedure which detects heme-protein complexes in polyacrylamide gels. Labeling of cell-surface polypeptides with 125I revealed that the 101-kDa heme-binding protein is expressed on the cell surface. Expression of the protein was regulated by growth temperature and was found to be encoded by the large virulence plasmid of S. flexneri. Deletion mutants and a TnS insertion mutant which were negative for Congo red binding (Crb-) did not express the 101-kDa protein. Enteroinvasive Escherichia coli strains that were Crb+, and whose plasmids shared homology with the S. flexneri virulence plasmid, also expressed the 101-kDa protein. Expression of the protein in S. flexneri and enteroinvasive E. coli correlated with the presence of a 9.2-kilobase EcoRI fragment of these plasmids.

The ability to bind the dye Congo red from agar medium is associated with virulence of Shigella species. DNA sequences conferring this property have been cloned from a large, 140-kilobase plasmid of Shigellaflexneri into a plasmid vector. This recombinant plasmid does not fully restore virulence to S. flexneri isolates which have lost the large plasmid. This indicates that other genes present on the 140-kilobase plasmid must also be required for virulence of S. flexneri. The cloned fragment contains a copy of the insertion sequence IS] closely linked to the gene for Congo red binding.