TOP DOWN MS/MS FOR POST TRANSLATIONAL MODIFICATION ANALYSIS OF TYPE IV PILI

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. For the gram negative bacterium Neisseria Meningitidis, host cell adhesion, bacterial aggregation and colony dissemination are key pathogenic stages moderated by type IV pili. These long, filamentous bacterial organelles are principally composed of the 17-kDa protein PilE, which is arranged in a helical fashion to create a strong and flexible polymer aggregate. PilE is known to carry several post-translational modifications (PTMs), e.g., a methylated N-terminus, cysteine bridge, glycerophos- phorylated serine and the disaccharide diacetamidotrideoxyhexose (DATDH). The DCMR lab, in collaboration with the group of Guillaume Dum¿nil, George Pompidou European Hospital, Paris, has recently demonstrated, using original top-down approaches by mass spectrometry, that half clinical isolates are alternatively modified with a previously uncharacterised monosaccharide, glyceramidotrideoxy- hexose (GATDH) [1]. During the course of this study, the presence of a minor form of pilE bearing two phosphoglycerol moieties led the DCMR lab to further investigate this modification. It appears that the expression of the enzyme that controls this modification is augmented upon contact of the bacterium with host cells. The most recent results localize the sites of the resultant additional phosphoglycerols and show unambiguously that these additional modifications disrupt pilus-pilus binding and thus decrease the aggregative properties of the bacteria. Complementary experiments show that these modifications are necessary for crossing of the epithelial layer, the first stage of pathogenesis. Taken together these results show for the first time that a PTM of type IV pili plays a crucial role in pathogenic virulence of Neisseria Meningitidis, a conclusion that obviously offers exciting prospects for similar pathogens bearing such pili [2]. During this project "top down" analysis of PilE was undertaken using ECD (Electron Capture Dissociation) on a 7-Tesla FT-ICR with promising results. The primary objective of the collaboration with BUSM Resource is to fully explore the potential of FT-ICR MS on a 12-T instrument, coupled with alternative activation techniques such as ETD, CID and IRMPD, and to evaluate ETD performed on an Orbitrap for "top down" PilE PTM characterisation. Development of these techniques to form a rapid method for PTM localization would enable a comprehensive of survey of PTM's across a wider number of Neisseria Meningitidis strains and ultimately other species bearing type IV pilli. [1] J. Chamot-Rooke, B. Rousseau, F. Lanternier, G. Mikaty, E. Mairey, C. Malosse, G. Bouchoux, V. Pelicic, L. Camoin, X. Nassif, G. Dumenil, , Proc. Nat. Acad. Sci. USA 104, 14783-14788 (2007) [2] J. Chamot-Rooke, G. Mikaty, C. Malosse, M. Soyer, A. Dumont, J. Gault, P. Martin, M. Trellet, G. Clary, P. Chaffey, L. Camoin, M. Nilges, X. Nassif, G. Dum¿nil, Science, (2011)