Phosphoproteome analysis of Borrelia burgdorferi




Nguyen, Trinh Thi-Duyen

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Borrelia burgdorferi is the causative agent of Lyme disease. This spirochetal pathogen alters it gene expression in response to environmental signals that are unique to the tick vector or mammalian hosts. Many of the genes that are up-regulated in response to mammalian host-specific signals such as temperature, pH, levels of dissolved oxygen and other uncharacterized factors are distributed in many linear and circular plasmids. Among these plasmids, the 54 kilo-base linear plasmid (lp54) encodes for the largest number of open reading frames (ORFs) that are up-regulated under mammalian host-specific conditions. While several whole genome transcriptional profile analysis provided a significant body of information regarding the levels of expression of these ORFs in response to various external signals, there is a dearth information regarding the proteomic changes exhibited by B. burgdorferi in response to these signals. We employed 2-dimensional gel electrophoresis in conjunction with selective labeling of phosphorylated proteins to identify B. burgdorferi proteins that exhibit differential levels of phophorylation in response to mammalian host-specific conditions (37°C and pH 6.8) in comparison to tick-specific conditions (23°C and pH 7.6). A total of 6 proteins were identified by mass spectrophotometric analyses using MALDI-TOF of which 4 (BB0337, BB0805, BB0502 and BB0445) were annotated as phosphoproteins. The identification of proteins that have been annotated as phosphoproteins validated the strategy to identify proteins that are a subset of the phosphoproteome of B. burgdorferi. In addition, BB0147 (borrelial flagellin, FlaB) and BBA41 (a hypothetical protein) were also identified as proteins with increased phosphorylation under mammalian host-specific conditions. In order to further characterize BBA41, encoded on lp54, we over-expressed BBA41 using the pET23a expression plasmid in E. coli with a 6X Histidine tag at the C-terminus and purified the recombinant BBA41(rBBA41) protein using nickel affinity column. Immunoblot analysis using either infection derived serum from C3H/HeN mice infected with virulent B. burgdorferi strain B31 or serum generated against borrelial lipoproteins showed reactivity to rBBA41 indicating that it is expressed under in vivo conditions. Mono-specific serum has been generated against BBA41 by immunizing female BALB/c mice with the purified rBBA41. In silico analysis of the BBA41 has not revealed a significant homology to proteins listed on a variety of databases. Inactivation of bba41 will provide insights into the role of this protein in the pathogenesis of B. burgdorferi in the mouse model of Lyme disease and provide additional clues as to the significance of its states of phosphorylation in pathogenic mechanisms of B. burgdorferi.


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Integrative Biology