000154128 001__ 154128
000154128 005__ 20190509132335.0
000154128 0247_ $$2doi$$a10.5075/epfl-thesis-4925
000154128 02470 $$2urn$$aurn:nbn:ch:bel-epfl-thesis4925-4
000154128 02471 $$2nebis$$a6196892
000154128 037__ $$aTHESIS
000154128 041__ $$aeng
000154128 088__ $$a4925
000154128 245__ $$aFolding and Structure of the Pore Forming Toxin Aerolysin
000154128 269__ $$a2011
000154128 260__ $$bEPFL$$c2011$$aLausanne
000154128 300__ $$a106
000154128 336__ $$aTheses
000154128 520__ $$aThe first obstacle encountered by a bacterial pathogen  once inside the host is the plasma membrane surrounding the  target cells. Throughout evolution bacteria has acquired and  maintained genes that upon stimulation express proteins  capable of damaging the membrane of other cells. Among these  proteins pore forming toxins (PFTs) are a major class of  bacterial effectors that are upregulated and secreted during  bacterial infections. As their name suggests, pore forming toxins are proteins  capable of inserting transmembrane pores in the membranes of  the target cells which in turn leads to the lysis of the cell  and release of nutrients. The mechanism by which the PFTs  function during a bacterial attack has been the subject of  extensive research over the years. In most cases PFTs are  produced by the bacteria as soluble proteins that require the  help of specialized secretion mechanisms to arrive as  functional proteins in the external milieu. Once secreted by  the producing bacteria these proteins diffuse towards the  target cell and bind to the target membrane. Once bound to  the plasma membrane of target cells they are capable of  initiating a series of structural changes that will  eventually lead to the conversion of the water-soluble PFT to  a membrane inserted channel. The series of events and the characterization of the  different structural changes required for a PFT to convert  from a water-soluble protein to a membrane inserted channel  is the subject of this thesis. Aerolysin, a PFT produced by  Aeromonas hydrophilla, is one of the best candidates for a  research into the details of the mode of action of bacterial  PFTs. This particular PFT is produced by the bacterium as a  soluble periplasmic protein and then secreted outside of the  bacterium as a fully folded protein with the help of a type  II secretion system. Binding to the target cell is achieved  through two high affinity binding sites that recognize sugar  modifications which are absent in A. hydrophila, a mechanism  that insures that the producing cell is not damaged by its  own PFT. Once bound to the target cell aerolysin requires  proteolytic activation, a step which cleaves a C-terminal  peptide (CTP). Activation is achieved using proteases present  on the target cell and the removal of the CTP is thought to  initiate the sequence of events leading to pore formation.  Following activation aerolysin is able to oligomerize forming  heptameric ring-like structures which spontaneously rearrange  forming a transmembrane beta-barrel through the membrane. My thesis project, focused on the structural changes  required in the mode of action of aerolysin, set off trying  to identify the aminoacid sequence involved in the formation  of the transmembrane beta-barrel. It was long thought that  aerolysin would cross the membrane in a porin like fashin,  forming a beta-barrel through the plasma membrane, primarily  due to the lack of a hydrophobic patch of aminoacids in its  sequence. An initial model proposed in the early '90s  postulated that the only region that could form the  transmembrane beta-barrel was the Domain 4 of the protein. In  this model the removal of the CTP in the activation process  would unravel the hydrophobic residues required for the  beta-barrel formation and insertion. We and others were able  to show however that the fourth domain of the protein is not  involved directly in the formation of the pore and we  identified a conserved loop in the third domain of the  protein which is responsible for the formation of the  beta-barrel. This loop presents an alternating pattern of  hydrophobic and hydrophilic residues, a requirement for the  formation of a transmembrane pore with a hydrophobic exterior  and a hydrophilic cavity. Our research led us to propose a  sequence of events upon insertion of the aerolysin pore in  which a rearrangement of the DIII-loops of the seven monomers  in the oligomer forms the initial beta-barrel and generates a  hydrophobic tip which drives insertion of the structure  through the membrane. Once the bilayer has been crossed the  hydrophobic tips folds back on the membrane in a rivet like  fashion, anchoring the pore. Following the identification of the DIII-loop as the  region that forms the transmembrane pore my researched  focused on the structural changes leading to the conversion  of a water-soluble protein to a membrane inserted oligomer.  While removal of the CTP is the key requirement for this  conversion, the role of the CTP in aerolysin mode of action  and the sequence of events triggered by its removal is not  fully understood. Using a combination of in vivo, in vitro  and in silico approaches we were able to show that the CTP  plays a wider role in the aerolysin mode of action than  previously thought. Indeed our research shows that the CTP is  initially required for the correct folding of the soluble  protein inside the bacterium, acting as a intramolecular  chaperone during the folding of aerolysin. Following folding  the CTP binds tightly to a hydrophobic pocket in the fourth  domain of the protein locking the PFT in its soluble  conformation, a role resembling C-terminal intramolecular  chaperones previously described for tail spikes of  bacteriophages or fiber forming collagen. This research will  be continued with a study on the structural changes triggered  by the removal of the CTP and their role in oligomerization  and pore formation. The main focus of my thesis project has been however the  determination of the structure of the oligomeric form of  aerolysin. This part of the project is still ongoing and will  be discussed in the final chapter of my thesis. Using 2D and  3D crystallography, AFM and modeling we hope to be able to  improve our current understanding of the aerolysin heptameric  form and the structural changes required in its  formation.
000154128 6531_ $$aaerolysin
000154128 6531_ $$apore forming toxins
000154128 6531_ $$aprotein structure
000154128 6531_ $$aprotein folding
000154128 6531_ $$amembrane damage
000154128 6531_ $$aaérolysine
000154128 6531_ $$atoxines formatrices de pores
000154128 6531_ $$astructure des protéines
000154128 6531_ $$arepliement des protéines
000154128 6531_ $$aalterations des membranes
000154128 700__ $$0242344$$g172742$$aIacovache, Mircea Ioan
000154128 720_2 $$aGoot Grunberg, Françoise Gisou van der$$edir.$$g171549$$0240085
000154128 8564_ $$uhttps://infoscience.epfl.ch/record/154128/files/EPFL_TH4925.pdf$$zTexte intégral / Full text$$s12470720$$yTexte intégral / Full text
000154128 909C0 $$xU11271$$0252037$$pVDG
000154128 909CO $$pthesis$$pthesis-bn2018$$pDOI$$ooai:infoscience.tind.io:154128$$qDOI2$$qGLOBAL_SET$$pSV
000154128 918__ $$dEDBB$$cGHI$$aSV
000154128 919__ $$aVDG
000154128 920__ $$b2011
000154128 970__ $$a4925/THESES
000154128 973__ $$sPUBLISHED$$aEPFL
000154128 980__ $$aTHESIS