A Salmonella enterica serovar Typhi gene that is selectively up-regulated upon bacterial invasion of eukaryotic cells was characterized. The open reading frame encodes a 298-amino-acid hydrophobic polypeptide (30.8 kDa), which is predicted to be an integral membrane protein with nine membrane-spanning domains. The protein is closely related (87 to 94% reliability) to different transport and permease systems. Gene expression under laboratory conditions was relatively weak; however, sevenfold induction was observed in a high-osmolarity medium (300 mM NaCl). The growth pattern in a laboratory medium of a serovar Typhi strain Ty2 derivative containing a 735-bp in-frame deletion in this gene, named gaiA (for gene activated intracellularly), was not affected. In contrast, the mutant was partially impaired in intracellular survival in murine peritoneal macrophages, as well as in human monocyte-derived macrophages. However, in the case of human macrophages, this survival defect was modest and evident only at late infection times (24 h). Despite the distinct intracellular survival kinetics displayed in macrophages of different species, the gaiA null mutant was significantly affected in its potential to trigger apoptosis in both murine and human macrophages. Provision of the gaiA gene in trans resulted in complementation of these phenotypes. Interestingly, the absence of a functional gaiA gene caused a marked attenuation in the mouse mucin model, as shown by the increase (3 orders of magnitude) in the 50% lethal dose of the mutant strain over that of the parental strain Ty2 (P < 0.05). Altogether, these data indicate that the product encoded by the gaiA gene is required for triggering apoptosis and bacterial survival within murine macrophages, which is consistent with the in vivo results obtained in the mouse mucin model. However, gaiA was not required for initial intracellular survival in human cells, indicating that its role in the natural host might be more complex than is suggested by the studies performed in the murine system.

A set of expression vectors was constructed which allows the expression of recombinant antigens under the control of Salmonella typhi promoters that are selectively activated after infection of eukaryotic cells. The pUC18Not derivatives contain a promoter downstream of the early transcriptional terminator from phage T7 and followed by a multiple cloning site, whereas the pBluescript II S/K derivatives contain the ribosomal RNA T 1 transcriptional terminator and also the strong translation signals of the Escherichia coli atpE gene. The expression cassettes are flanked by NotI or PacI sites to simplify their subcloning where required. The resulting vectors were validated using the S1 subunit of pertussis toxin as a model antigen and Salmonella typhimurium aroA SL7207 as a carrier. The S1 subunit was efficiently expressed by recombinant Salmonella within Henle 407 cells. ß

A set of expression vectors was constructed which allows the expression of recombinant antigens under the control of Salmonella typhi promoters that are selectively activated after infection of eukaryotic cells. The pUC18Not derivatives contain a promoter downstream of the early transcriptional terminator from phage T7 and followed by a multiple cloning site, whereas the pBluescript II S/K derivatives contain the ribosomal RNA T(1) transcriptional terminator and also the strong translation signals of the Escherichia coli atpE gene. The expression cassettes are flanked by NotI or PacI sites to simplify their subcloning where required. The resulting vectors were validated using the S1 subunit of pertussis toxin as a model antigen and Salmonella typhimurium aroA SL7207 as a carrier. The S1 subunit was efficiently expressed by recombinant Salmonella within Henle 407 cells.