Project description:Secretion of outer membrane vesicles (OMV) presents an important phenomenon in Gram-negative bacteria and they play multiple roles in their lifestyle including virulence and host-pathogen interaction. Francisella tularensis secretes unusually shaped tubular OMV filled with immunoreactive material and virulence factors. Mice were immunized with OMV isolated from Francisella tularensis subsp. holarctica, fully virulent strain FSC200. Their sera were then used for detection of immunoreactive proteins.
Project description:Comparison of enriched membrane fractions of Francisella tularensis subsp. holarctica strain FSC200 and its DsbA mutant by SILAC analysis.
Project description:Secretion of outer membrane vesicles (OMV) presents an important phenomenon in Gram-negative bacteria and they play multiple roles in their lifestyle including virulence and host-pathogen interaction. Francisella tularensis secretes unusually shaped tubular OMV. We here present a proteomic comparison of whole cell lysates, OMV and membrane fractions derived from two F. tularensis strains: a moderately virulent subsp. holarctica strain FSC200 and highly virulent subsp. tularensis SchuS4 strain. We describe a novel approach to the cross-species proteomic comparison based on creating an intersection protein database from the individual single-species databases. This approach proved to be less prone to quantification errors arising from differences in the protein sequences than using the individual databases. The consecutive comparison of the subproteomes of OMV and membranes of the two strains allowed us to distinguish the differences in protein amounts caused by global expression changes from those caused by preferential protein packing to OMV or membrane. Amongst the most different proteins we detected proteins involved in the biosynthesis and metabolism of the bacterial envelope components like O-antigen, lipid A, phospholipids, and fatty acids, as well as some of the major structural outer membrane proteins.
Project description:Francisella tularensis is one of three bacterial species designated as a Category A select agent by the Centre for Disease Control (CDC), a category indicating agents most likely to be employed as a biological weapon. F. tularensis can be divided into four different subspecies, and it is well known that the type, severity and duration of the disease can differ substantially depending on what subspecies is responsible for the infection. Of the four subspecies, subsp. tularensis (Type A) and subsp. holartica (Type B) are of primary clinical significance, and account for nearly all recorded incidences of the disease in humans. Though Type A is considered to be more virulent than Type B, recent reports have shown that Type A can be further sub-divided into two genetically distinct populations, termed A.I and A.II, which differ with respect to geographical location, disease outcome and source of recovered isolates. Of these two subpopulations, clinical data suggests that Type A.I strains are significantly more virulent than Type A.II, and Type A.II strains appear to have a disease outcome similar to infections with Type B. During natural infections, host mononuclear phagocytes appear to be the primary target of all F. tularensis subsp. Despite the differences in disease outcome between different subspecies, the mechanisms involved in phagosomal escape, the modulation of phagosomal biogenesis, phagosomal disruption and bacterial egress appears to be indistinguishable between subspecies, at least at a physiological level. In collaboration with Dr. Patrick McGann at Walter Reed Army Institute of Research (WRAIR) we have been studying the differential gene expression of F. tularensis during macrophage infection. Dr. McGann provided the PFGRC with RNA samples from F. tularensis strains LVS and Shuh4 isolated from infected macrophages. Samples were interrogated using high throughput qRT-PCR using 1,067 primer pairs.