Abstract
Francisella tularensis, the causative agent of the disease tularaemia, is one of the most infectious bacteria known. F. tularensis subspecies tularensis (type A) and holarctica (type B) are the major subspecies, with the former being highly virulent for humans (Conlan et al., 2002). A live vaccine strain (LVS) of F. tularensis empirically derived from a virulent strain of type B F. tularensis provides considerable protection against highly virulent type A strains in humans (Conlan et al., 2002; Fulop et al., 2001). Although the F. tularensis LVS is attenuated for humans, it is fully virulent for mice (Conlan et al., 2002), and infection with F. tularensis LVS in mice has been used as an experimental model of tularaemia in a number of studies.
Immunization of mice with LPS derived from F. tularensis LVS induced protection against intraperitoneal challenge with the LVS but not against a virulent strain of type A F. tularensis. However, the immunization significantly increased the survival time in mice challenged with the virulent strain of type A F. tularensis (Fulop et al., 2001). Similarly, mice vaccinated with O-antigen of the LPS from F. tularensis LVS chemically conjugated to BSA were protected against an intradermal challenge with a highly virulent strain of type B F. tularensis but not against a virulent type A strain (Conlan et al., 2002). mAbs directed against O-antigen and core polysaccharide of the LPS from F. tularensis LVS recognized both type A and type B strains (Fulop et al., 1991). This suggests the presence of common epitopes in the LPS of both subspecies. However, the mouse protection studies suggest possible differences in the structure of LPS/O-antigens between the two subspecies (Fulop et al., 2001; Prior et al., 2003). Conversely, different mechanisms of protection may be required to resolve infections caused by the two subspecies (Fulop et al., 2001).
The O-antigen structure of F. tularensis strain 15 (a vaccine strain derived from type B F. tularensis in the former Soviet Union) was determined to contain repeating tetra-saccharide subunits, 4-(α-D-GalpNAcAN-(1-4)-α-D-GalpNAcAN-(1-3)-ß-D-QuipNAc-(1-2)-ß-D-Quip4NFo-1), using 1H- and 13C-NMR spectroscopy (Vinogradov et al., 1991). Francisella tularensis LVS was also found to express O-antigen identical to that of F. tularensis strain 15 (Conlan et al., 2002). Studies on the structure of O-antigen from virulent strains of type A F. tularensis have been limited to the Schu S4 strain, which is a virulent but highly passaged laboratory strain (Prior et al., 2003). The repeating units of the O-antigens from the Schu S4 strain and the LVS were presumed to be the same based on MALDI (matrix-assisted laser desorption/ionization)-MS analysis (Prior et al., 2003). In the present study we report the 1H- and 13C-NMR spectroscopy structural analysis of O-antigen from a field strain of type A F. tularensis (strain OSU 10).
F. tularensis subspecies tularensis strain OSU 10 was isolated from a cat that died of tularaemia and subspecies identification was based on Biolog metabolic fingerprinting (Biolog) and PCR (Petersen et al., 2004) and confirmed by the Centers for Disease Control (Atlanta, GA, USA). LPS from strain OSU 10 was isolated using the Tri-reagent method described elsewhere (Yi & Hackett, 2000), and analysed by SDS-PAGE and Western blotting. LPS samples were run on 12 % acrylamide gels followed by silver staining. Isolated LPS showed the characteristic ladder pattern on acrylamide gels, indicating variation in the polysaccharide side-chain length (Fig. 1a). Further, the LPS was reactive with mAbs specific for F. tularensis O-antigen (BioDesign; Fig. 1b).