INTRACELLULAR MULTIPLICATION AND VIRULENCE OF SHIGELLA-FLEXNERI AUXOTROPHIC MUTANTS

We have constructed and analyzed a group of Shigella flexneri 5 auxotr ophic mutants, The wild-type strain M90T was mutagenized in genes enco ding enzymes involved in the synthesis of (i) aromatic amino acids, (i i) nucleotides, and (iii) diaminopimelic acid, In this way, strains wi th single (aroB, aroC, aroD, purE, thyA, and dapB) and double (purE ar oB, purE aroC, purE aroD, purE thyA) mutations were obtained, Although the Aro mutants had the same nutritional requirements when grown in l aboratory media, they showed different degrees of virulence in vitro a nd in vivo, The aroB mutant was not significantly attenuated, whereas both the aroC and aroD strains were severely attenuated, p-Aminobenzoi c acid (PABA) appeared to be the main requirement for the Aro mutants' growth in tissue culture, Concerning nucleotides, thymine reduced the pathogenicity, whereas adenine did not. However, when combined with a nother virulence-affecting mutation, adenine auxotrophy appeared to po tentiate that mutation's effects, Consequently, the association of eit her the purE and aroC or the purE and aroD mutations had a great effec t on virulence as measured by the Sereny test, whereas the purE aroB d ouble mutation appeared to have only a small effect, All mutants excep t the dapB strain seemed to move within a Caco-2 cell monolayer after 3 h of infection, Nevertheless, the auxotrophs showing a high intracel lular generation time were negative in the plaque assay, Knowledge of each mutation's role in attenuating Shigella strains will provide usef ul tools in designing vaccine candidates.