We report here that the Shigella invasion plasmid antigen (Ipa)B, which is sufficient to induce apoptosis in macrophages, binds to caspase (Casp)-1, but not to Casp-2 or Casp-3. Casp-1 is activated and its specific substrate interleukin-1␤ is cleaved shortly after Shigella infection. Macrophages isolated from Casp-1 knock-out mice are not susceptible to Shigella-induced apoptosis, although they respond normally to other apoptotic stimuli. Shigella kills macrophages from casp-3, casp-11, and p53 knock-out mice as well as macrophages overexpressing Bcl-2. We propose that Shigella induces apoptosis by directly activating Casp-1 through IpaB, bypassing signal transduction events and caspases upstream of Casp-1. Taken together these data indicate that Shigella-induced apoptosis is distinct from other forms of apoptosis and seems uniquely dependent on Casp-1.

Caspases are intracellular proteases that mediate mammalian cell apoptosis. Caspase-1 (Casp-1) is a unique caspase because it activates the proinflammatory cytokines interleukin (IL)-1beta and IL-18. Shigella flexneri, the etiological agent of bacillary dysentery, induces macrophage apoptosis, which requires Casp-1 and results in the release of mature IL-1beta and IL-18. Here we show that casp-1(-/-) mice infected with S. flexneri do not develop the acute inflammation characteristic of shigellosis and are unable to resolve the bacterial infection. Using casp-1(-/-) mice supplemented with recombinant cytokines and experiments with IL-1beta(-/-) and IL-18(-/-) mice, we show that IL-1beta and IL-18 are both required to mediate inflammation in S. flexneri infections. Together, these data demonstrate the importance of Casp-1 in acute inflammation and show the different roles of its substrates, IL-1beta and IL-18, in this response.

We have previously reported that the presence of a 180-kilobase plasmid encoding production of aerobactin was correlated with the virulence of Kkebsiella pneumoniae Kl and K2 isolates. This work demonstrates that a variant of a K2 strain which has lost this plasmid, pKPlOO, becomes avirulent. Labeling of this plasmid with the mobilizable, replication-defective element pME28, used here as a mobilizable transposon, allowed the transfer of this plasmid into a plasmidless derivative. Virulence was restored upon reacquisition of this tagged plasmid, pKP1O1. In addition to aerobactin production, another phenotype could be correlated with the presence of this virulence plasmid: the mucoid phenotype of the bacterial colonies. Both wild-type and plasmidless strains are encapsulated, but only the former presented mucoid colonies. Participation of this phenotype in the virulence of K. pneumoniae was demonstrated by constructing a mutant altered in the plasmid gene encoding this phenotype. The resulting strain demonstrated a 1,000-fold decrease in virulence. Introduction of the recombinant plasmid pKP200 carrying the gene encoding this mucoid phenotype into Escherichia coli HB101 also led to the production of a mucoid phenotype. Rocket immunoelectrophoresis demonstrated that in E. coli this phenotype was due to the production of colanic acid. On the other hand, neither the overproduction of K2 capsular polysaccharide nor the presence of colanic acid was detected in mucoid strains of K. pneumoniae. We conclude that this mucoid phenotype is definitely an important virulence factor of K. pneumoniae. It is due to the plasmid-encoded production of a substance which is different from colanic acid and the capsular polysaccharide of K. pneumoniae.