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

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 对于革兰氏阴性细菌脑膜炎奈瑟氏菌，宿主细胞粘附、细菌聚集和菌落散布是由IV型皮利调节的关键致病阶段。这些长的丝状细菌细胞器主要由17-kDa蛋白PilE组成，其以螺旋方式排列以产生坚固且柔性的聚合物聚集体。已知PilE携带几种翻译后修饰（PTM），例如，甲基化N-末端、半胱氨酸桥、甘油磷酸化丝氨酸和二糖二乙酰氨基三脱氧己糖（DATDH）。DCMR实验室与巴黎乔治蓬皮杜欧洲医院的Guillaume Dum <$nil小组合作，最近通过质谱法使用原始的自上而下方法证明，一半临床分离株被先前未表征的单糖甘油酰胺三脱氧己糖（GATDH）替代修饰[1]。 在本研究过程中，存在携带两个磷酸甘油部分的次要形式的pilE，导致DCMR实验室进一步研究这种修饰。看来控制这种修饰的酶的表达在细菌与宿主细胞接触时增强。最新的结果本地化的网站所产生的额外的磷酸甘油，并明确显示，这些额外的修改破坏菌毛菌毛结合，从而降低细菌的聚集特性。补充实验表明，这些修改是必要的跨越上皮层，发病的第一阶段。综上所述，这些结果首次表明IV型皮利的PTM在脑膜炎奈瑟氏球菌的致病性毒力中起关键作用，这一结论显然为携带这种皮利的类似病原体提供了令人兴奋的前景[2]。在该项目期间，在7特斯拉FT-ICR上使用ECD（电子捕获解离）进行了PilE的“自上而下”分析，结果令人满意。与BUSM Resource合作的主要目的是充分探索FT-ICR MS在12 T仪器上的潜力，以及替代活化技术，如ETD，CID和IRMPD，并评估在Orbitrap上进行的ETD，以进行“自上而下”的PilE PTM表征。开发这些技术以形成PTM定位的快速方法将使得能够在更广泛数量的脑膜炎奈瑟菌菌株和最终携带IV型菌毛的其他物种中全面调查PTM。 [1]J. Chamot-Rooke，B. Rousseau，F. Lanternier，G. Mikaty，E.迈雷角马洛斯湾Bouchoux，V. Pelicic，L.卡莫因，X.纳西夫湾Dumenil，，Proc. Nat. Acad. Sci. USA 104，14783-14788（2007） [2]J. Chamot-Rooke，G.米卡蒂角马洛斯湾Soyer，A. Dumont，J. Gault，P. Martin，M.特雷莱湾Clary，P. Chaffey，L. Camoin，M.尼尔格斯岛纳西夫湾Dum <$nil，Science，（2011）

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)