Fig. 8. Salmonella typhi CdtB is not a substrate of the type III secretion systems (TTSSs). The absence of CdtA and CdtC, coupled with the requirement for CdtB to be delivered into the cytosol to reach its site of activity, raised the possibility that this protein may be a substrate of a TTSS (1, 2). S. typhi encodes two TTSSs, one within pathogenicity island 1 (SPI-1 TTSS), which is required for bacterial internalization, and another within pathogenicity island 2 (SPI-2 TTSS), which is required for systemic infection (3). When grown in vitro, Salmonella expresses only the SPI-1 TTSS, because the SPI-2 TTSS is expressed only when the bacteria has reached the intracellular environment (4). Because cdtB is expressed only on bacterial internalization, we first tested whether a S. typhi mutant defective in the SPI-2 TTSS retained cytotoxicity. As shown in A, the SPI-2-defective S. typhi spiA mutant strain showed identical toxicity to the wild type (w. t.), indicating that this protein secretion system is not required for CDT activity. Because CDT toxicity requires bacterial internalization, an equivalent experiment with a SPI-1-defective strain was not feasible. However, because the SPI TTSS is expressed in vitro, we tested whether CdtB could be secreted into the culture supernatant in a SPI-1 TTSS-dependent manner when expressed from an inducible promoter (p_araBAD). Wild-type and SPI-1 TTSS-deficient (invA) strains of S. typhi carrying a plasmid encoding an epitope-tagged CdtB expressed from an inducible promoter were grown under conditions that stimulate SPI-1 TTSS gene expression, and culture supernatants were examined for the presence of CdtB. As shown in B, CdtB was equally detected (≈20% of total protein) in culture supernatants of both wild-type and invA S. typhi strains. The presence of CdtB in the supernatant was not due to bacterial lysis, because type III secreted proteins were found only in the supernatant of the wild-type strain, and no significant amount of the cytoplasmic protein 6-phosphogluconate dehydrogenase was detected in either strain. Furthermore, the secreted form of CdtB was of lower molecular weight than the species observed in whole-cell lysates. Because the CdtB protein carries the epitope tagged at the carboxyl terminus, the smaller CdtB likely represents the processed form with a cleaved sec signal sequence. These results indicate that CdtB is not a substrate of either the SPI-1 or SPI-2 TTSS. The presence of a predicted sec-dependent signal sequence in CdtB is in keeping with these observations, because substrates of TTSS do not possess this signal sequence. In addition, these results showed that CdtB is secreted into culture supernatants by an as-yet-unidentified secretion system.

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