Inactivation of dsbA, but not dsbC and dsbD, affects the intracellular survival and virulence of Shigella flexneri

In this study, three mutants, dsbA::kan, dsbC-kan, and dsbD-kan, of Shigella flexneri serotype 5 were constructed and characterized to investigate the role of the periplasmic thiol:disulfide oxidoreductases in pathogenicity. In gentamicin protection assays and the Sereny test, the dsbA mutant showed reduced virulence while the dsbC and dsbD mutants were similar to the wild type. That inactivation of dsbA was responsible for the reduced virulence was verified by complementation with the cloned wild-type gene in in vitro and in vivo assays. Despite the changed virulence behavior, the dsbA mutant could penetrate HeLa cells 15 min postinfection, consistent with the fact that it actively secretes Ipa proteins upon Congo red induction. Furthermore, the dsbA mutant was able to produce actin comets and protrusions, indicating its capacity for intra-and intercellular spread. However, a kinetic analysis of intracellular growth showed that the dsbA mutant barely grew in HeLa cells during a 4-h infection whereas the wild type had a doubling time of 41 min. Electron microscopy analysis revealed that dsbA mutant bacteria were trapped in protrusion-derived vacuoles surrounded by double membranes, resembling an icsB mutant reported previously, Moreover, the trapped bacteria appeared to be lysed simultaneously with the double membranes, resulting in characteristic empty vacuoles in the host cell cytosol, Thus, the attenuation mechanism for dsbA mutant appears to be more complicated than was previously suggested.