Eric Durand Molecular Microbiology and Pathogenicity in Pseudomonads/Alain Filloux. Web site:
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1 seudopilus ssembly by the ype II Secretion Machinery of seudomonas aeruginosa: rchitecture, Structure of subunits, Functions and Biogenesis Control Eric Durand Molecular Microbiology and athogenicity in seudomonads/lain Filloux Web site: ctive enzymes/ toxines released in the extracellular medium he seudomonas aeruginosa ype II Secretion athway: the Xcp Secreton ECM Step 2 OM - eriplasmic intermediate - Folding step - Secretion signal recognition Step 1 Sec Xcp secreton at IM Cytoplsamic precursor with an N-ter signal sequence. S Unfolded state D + i RR Folded state C 1
2 Similarities between the Xcp SECREON and Components of the Xcp SECREON the il SYSEM Xcp Secreton Extracellular medium Exoproteins il System Existence of the pseudopilus ype IV pili rchitecture of the pseudopilus ily1 Secretin Q SECREINS Secretin ilq OM Open V Closed Z seudopilins/ilins Cytoplasm X S Nase R Y IMs eptidase Xcp O /ild Nases W X V U ilc Nase ilb Nase il e il IM ssembly Retraction seudopilins/ilins Relative amounts: Xcp G ;-U H ;-V I ; -W J ; -X K il 1/ he major pseudopilin Xcp is able to assemble multifibrillar structure: he ype II seudopilus. 2
3 L exposition de la pseudopiline Xcp à la surface cellulaire dépend de sa surproduction ped (xcp) Cellules Fractions sheared Microscopie Electronique O1 = W O1xcp - Immunoblot révélant seulement la présence de la protéine Xcp (α) La pseudopiline Xcp (16,2kD) est retrouvée dans la fraction «sheared» seulement en condition de surproduction. La flagelline FliC (49,24kD) et la piline il (15,5kD) sont également présentes dans ces mêmes fractions (pistes 4-6), mais non détectées ici. Bacteria overproducing Xcp G are covered with extracellular pilus-like appendages. ransmission Electron Microscopy, Negative Staining Bactéries entières cultures sur boîtes Individual fibrils «Bundle» Single filament thickness =7-9nm; Bundles thickness nm; lenght= up to 10µm. 3
4 Xcp G is assembled in a multifibrillar structure called he ype II seudopilus O1/pED EM analysis, Immunogold labelling anti-xcp G Bactéries entières cultures sur boîtes Flagella ype II pseudopilus ype IV pilus SEUDOILI RERION (IMα) Kpil-/fliC- /ped 4
5 ISIL IMF Bacterium Isolated pseudopili nchored pseudopili ISIL: In Situ Immunogold Labelling. echnique vue dans Interactions Bactéries phytopathogènes/hôtes, HR pili de seudomonas seringue. 2/ Ultrastructural studies of the pseudopilus. Structural studies of the pseudopilins. 5
6 Hypothetical ype II pseudopilus substructure ype II pseudopilus substructure - wisted bundle - Helical fibrils - Longitudinal repeat of 2 Xcp subunits - Held by side projections Original Image Repeat unit Fourier ransform Enlargement 4.3 nm 3.5 nm Filtered image Side projection Image Filtering James Sturgis (LISM) Central axis First assay of cryo-electron omography and 3D reconstruction 2D images 3D reconstruction( em program ) 3D reconstruction/surface-rendered ( mira viewer ) Goal: o obtain a 3D model of the architecture of the single filaments forming the type II pseudopilus. (r. W. Baumeister / Julia Küerner / Max-lanck-Institut Munich ) 6
7 Single article nalysis: First Steps owards a 3D model of the pseudopilus lain Bernadac (IBSM) - Logiciel: EMN (3 modules: Eman, Boxer, Ctfit ) 2D images/cryoem - Selection of pseudopili fragments - Fragments have been straightened -andcentered (module «Boxer») - Starting matrix = 323 images - Images were sorted out - Classes were defined - Class selection Modelisation: starting model (Software: «Modelex») Further steps: (i) Correction of image defects (module «Ctfit»), (ii) Refine the starting model (Refine/EMN) Improvement: (i) Increase contrast of the 2D images, (ii) Greatly increase the number of images in the starting matrix. type II pseudopilus in the Xcp Secreton. What is it useful for? Close the secretin pore Biofilm formation ump the exoprotein through the secretin pore MEC ME C Q D exoprotein Q D MI Y L Z M C Chromosomique expression S F R E Xcp G ;-U H ;-V I ; -W J ; -X K pseudopilins Xcp O /ild Xcp overproduction 7
8 WH MKES HEM SO DHEREN? Its spider s web Its pseudopilus? Quickime et un décompresseur GIF sont requis pour visualiser cette image. 1/ Molecular piston hypothesis a) Q OM b) c) X IM S S ore closed () disassembled (X) [X>] C Substrate arrival he Xcp pool displaces XcpX elongation proceeds [>X] reaches the OM ore opening/substrate secretion XcpX comes back=elongation arrest [>X] d) Xcp conformational changes (X) Xcp degradation disassembly a) [X>] 8
![1/ Molecular piston hypothesis a) Q OM b) c) X IM S S ore closed () disassembled (X) [X>] C Substrate arrival he](/docs-images/58/4618656/images/page_8.jpg "Xcp pool displaces XcpX elongation proceeds [>X] reaches the OM ore opening/substrate secretion XcpX comes")
9 2/ Secretin plug hypothesis a) b) c) 1 pp transpéri. =10xXcp = 5x repeat units ore closed assembled Substrate arrival XcpX incorporation XcpX induces Xcp conformational changes Xcp degradation disassembly/ore opening Substrate secretion d) Xcp pool displaces XcpX and/or XcpX degradation assembly ore closing 9
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