O-antigen expression analyses.

Slide agglutination reactions were performed with the following serotype-specific rabbit polyclonal antisera: S. flexneri serotype 1, 2, 3, 4, 5, and 6 antisera, S. flexneri serotype II-specific antiserum, S. flexneri 3,4 epitope-specific antiserum, S. flexneri serotype III-specific antiserum, S. flexneri 6 epitope-specific antiserum, S. flexneri 7,8 epitope-specific antiserum, or S. Typhi O-specific 9 factor antiserum (Difco). For immunoblotting, Salmonella, Shigella, and E. coli strains with or without various recombinant plasmids were grown overnight with aeration at 37°C in medium containing appropriate antibiotics, and the bacterial cultures were standardized by optical density. LPS was purified using an LPS extraction kit (Boca Scientific, FL), according to the manufacturer's instructions. Purified LPS was separated by 14% Tris-glycine PAGE gels (Life Technologies, NY). Standard Western blotting procedures were carried out with the above-mentioned antibodies for the identification of specific LPS types.

The stability of recombinant clones of Ty21a-2a, Ty21a-2al, Ty21a-2aol, and Ty21a-3a (i.e., expressing S. flexneri 2a or 3a O-antigens) was tested by immunoblotting of colonies plated from an overnight culture (i.e., 24-h culture following high dilution = ∼25 generations). Subsequent similar serial dilution and regrowth were conducted, for a total of ∼75 generations. The resulting colonies from agar platings were transferred to a nitrocellulose membrane and analyzed by standard Western blotting procedures using the LPS-specific antibodies specified above. More than 300 colonies were examined from each strain after ∼75 generations of growth to assess genetic stability.