Proteomic dissection of biofilms and identification of T-cell antigens of Bordetella pertussis

Pertussis (whooping cough) is a respiratory disease caused by Bordetella pertussis. The currently used vaccine provides a relatively short duration of immunity and pathogen adaption against the vaccine has been linked to a resurgence of pertussis globally. Past research and vaccine development has been based on planktonic cells, however, recent studies have shown that B. pertussis readily forms biofilms. Biofilms are a community of cells encased within a matrix and these cells have been shown to be more resilient against antimicrobials. Studies of the biofilm lifestyle may provide further insight into B. pertussis evolution and pathogenesis. A comparative study of biofilm and planktonic cells was performed by combining mass spectrometry (MS) and metabolic modelling. It was found that the glyoxylate shunt was utilised in planktonic cells while the biofilm cells completed the tricarboxylic acid cycle providing novel targets for biofilm disruption. Single nucleotide polymorphism (SNP) cluster I (ptxP3/prn2) strains that have overtaken the previously dominant SNP cluster II (ptxP1/prn3) strains were compared in biofilm conditions. Denser biofilm structures for a cluster I strain were identified using confocal laser scanning microscopy. MS analysis revealed cytochrome proteins were strongly upregulated in the SNP cluster I strain, however, this change was specific to the individual strain. Interestingly, increased biofilm formation was found when the strains were grown in low oxygen, which are conditions found during infection. The cluster II strains had increased levels of cells dispersed from the biofilm compared to the cluster I strains in these conditions. These results show that there is an underlying difference in the way the clusters respond to changes to oxygen concentrations when grown in biofilm conditions and may relate to the dominance of the cluster I strains. Finally, an immunoproteomic method for identifying novel vaccine antigens was optimised. This method utilised immortalised mouse dendritic cells ...