Francisella tularensis is the causative agent of the zoonotic disease tularemia. Four subspecies of this pathogen, namely ssp. tularensis, mediaasiatica, holarctica, and novicida are spread throughout the northern hemisphere. Although there are marked variations in their virulence to mammals, the subspecies are difficult to identify as they are closely genetically related. We carried out the comparative proteome analysis of cellular extracts from isolates representing the highly virulent subspecies tularensis, and the less virulent subspecies mediaasiatica and holarctica in order to identify new diagnostic markers and putative factors of virulence. We identified 27 protein spots that were either specifically present or at significantly higher abundance in ssp. tularensis strains, 22 proteins in ssp. mediaasiatica strains, and 26 proteins in ssp. holarctica strains. Subspecies tularensis-specific proteins might represent putative virulence factors. Of 27 identified tularensis-specific spots 17 represented charge and mass variants of proteins occurring in other subspecies, 7 spots were found to be present at higher abundance, and 3 spots were specifically present in tularensis strains. Amongst them, PilP protein, as a component necessary for the biogenesis of the type IV pilus, virulence and adhesion factor for many human pathogen, was identified. Furthermore, the identification of additional 27 proteins common for ssp. tularensis and mediaasiatica, and 19 proteins shared by ssp. mediaasiatica and holarctica documented apparent closer genetic similarity between ssp. tularensis and mediaasiatica.
The whole cell lysate of Coxiella burnetii strain RSA 493 was separated by two-dimensional electrophoresis and more than 500 protein spots were found on silver-stained reference map. Spots from the gels were subjected to identification based on peptide mass fingerprinting (PMF). In order to identify additional proteins, tandem mass spectrometry (MS/MS) using electrospray and matrix-assisted laser desorption/ionization techniques was applied. The three independent approaches resulted in the identification of 197 open reading frames (ORFs). Fifty-two proteins were identified by PMF and at least with one of the MS/MS methods, 37 proteins with both MS/MS instruments, and 19 proteins with all three techniques applied. All predicted C. burnetii ORFs were compared with the Clusters of Orthologous Groups database. The data related to identified proteins were stored and indexed in a file that can be read and searched using Microsoft Access.
Proteome analysis of Gram-negative facultative intracellular pathogen Francisella tularensis (F. tularensis) live vaccine strain has been performed only on whole-cell extracts so far. This is the first study dealing with the analysis of the membrane subproteome of this microorganism. A fraction enriched in membrane proteins obtained by carbonate extraction was separated using two-dimensional electrophoresis and all visualized spots were identified by mass spectrometry. The reference map is the basis for further comparative analyses of virulent and non-virulent F. tularensis strains.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.