Figure 2

Proteomic technologies used in applied research on C. burnetii pathogenesis. Several proteomic approaches have been used to identify biomarkers of Q fever or to characterize the proteome of C. burnetii. (a) Laboratory pipeline for biotyping. Biotyping of C. burnetii is not yet applied routinely because of restrictions in manipulating the organism, which is a potential bioterrorism agent. With the recent development of axenic solid culture for C. burnetii, however, the laboratory isolation of strains from blood- culture-negative samples associated with endocarditis (chronic Q fever) or from a variety of other samples such as blood or rhinopharyngeal swabs (acute Q fever) might be possible. The sample or bacterial products are subjected to analysis by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS). Colonies of C. burnetii are picked from solid medium, co-crystallized with matrix, and processed for MALDI TOF MS analysis. The obtained MS spectra are analyzed against an available database, which allows identification of the bacteria. In parallel, classical phenotypic identification methods, including Gimenez- or Gram-staining of bacteria and biochemical tests, can be applied to confirm the identity of bacteria. (b) Laboratory pipeline for immunoproteomics. The whole-cell protein extract or fraction (such as sarcosyl-insoluble fraction, containing mainly membrane proteins) is resolved on two-dimensional (2D) acrylamide gels. The resolved proteins are stained (using silver nitrate or Coomassie blue) or transferred onto nitrocellulose or polyvinylidene fluoride (PVDF) membranes and then processed for immunoblotting with sera. The sera are from patients or animals with Q fever (general, acute or chronic Q fever) and from naïve subjects (control group). The immunoblots are analyzed and compared to silver-stained gels (using commercially available software). This analysis can be improved by statistical methods (such as principal component analysis (PCA)), which allows more discriminating spot selection. All the selected spots are subjected to MS identification. In some studies, the best targets are validated by using different approaches, such as recombinant-based enzyme-linked immunosorbent assay (ELISA) or protein array. (c) Laboratory pipeline for recombinant protein-based approaches. The large-scale recombinant protein systems allow a high level of genome coverage (>75% of predicted open reading frames (ORFs)). C. burnetii proteins are expressed using Escherichia coli or acellular translation systems (such as the rapid translation system (RTS)). Expressed and/or purified recombinant proteins are transferred to arrays and screened with serum samples from patients and control subjects. Seroreactivity is detected using a fluorescently labeled anti-human IgG antibody. The arrays are read using a laser confocal scanner and the signal intensity of each protein is quantified. The results are analyzed and normalized using statistical tools. The normalized intensity is shown according to a color scale. The top seroreactive proteins are selected by using a determined cut-off.